Location: Plant Science ResearchTitle: A CRISPR/dCas9 toolkit for functional analysis of maize genes
|GENTZEL, IRENE - The Ohio State University|
|PARK, CHAN HO - The Ohio State University|
|BELLIZZI, MARIA - The Ohio State University|
|XIAO, GUIQING - The Ohio State University|
|GADHAVE, KIRAN - North Carolina State University|
|MURPHREE, COLIN - North Carolina State University|
|YANG, QIN - North Carolina State University|
|LAMANTIA, JONATHAN - The Ohio State University|
|REDINBAUGH, MARGARET - The Ohio State University|
|SIT, TIM - North Carolina State University|
|WANG, GUO-LIANG - The Ohio State University|
Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2020
Publication Date: 1/1/2021
Citation: Gentzel, I.N., Park, C., Bellizzi, M., Xiao, G., Gadhave, K.R., Murphree, C., Yang, Q., Lamantia, J., Redinbaugh, M.G., Balint Kurti, P.J., Sit, T.L., Wang, G. 2021. A CRISPR/dCas9 toolkit for functional analysis of maize genes. Plant Methods. https://doi.org/10.1186/s13007-020-00675-5.
Interpretive Summary: This work describes methods for introducing machinery for gene editing and manipulation into single-celled maize protoplasts.
Technical Abstract: Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for manipulating gene expression 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.