A CRISPR/dCas9 toolkit for functional analysis of maize genes

Area of Science:

• Plant Genomics
• Molecular Biology
• Biotechnology

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is a key tool in plant functional genomics.
• Deactivated Cas9 (dCas9) enables manipulation of gene expression, distinct from its gene-editing capabilities.

Purpose of the Study:

• To develop a vector toolkit for analyzing dCas9-mediated gene expression modulation in maize protoplasts.
• To establish improved methods for maize protoplast isolation and transfection for enhanced experimental efficiency.

Main Methods:

• Development of dCas9 vectors for gene expression activation (CRISPRa) and repression (CRISPRi) in maize.
• Optimization of maize protoplast isolation and transfection protocols.
• Utilizing the toolkit to analyze guide RNA (gRNA) candidates for gene regulation.

Main Results:

• A functional vector toolkit for CRISPR-mediated gene expression manipulation in maize protoplasts was created.
• An improved protocol for maize protoplast isolation and transfection was successfully implemented.
• Demonstrated utility of the toolkit for enhancing or repressing maize gene expression.

Conclusions:

• The developed maize protoplast toolkit is expected to accelerate the analysis of gRNA candidates.
• This resource will facilitate genetic studies of important trait genes in maize, a recalcitrant species for transformation.
• Streamlines functional genomics research in maize.