An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants

Area of Science:

• Plant molecular biology
• Gene editing technologies
• Agricultural science

Background:

• CRISPR-Cas9 is widely used for microRNA (miRNA) gene knockout in plants, but its small insertions/deletions limit complete gene disruption.
• Complete knockout of miRNA genes is crucial for understanding their regulatory roles in plant development and stress responses.

Purpose of the Study:

• To evaluate CRISPR-Cas12a as a superior tool for generating complete miRNA gene knockouts in rice compared to CRISPR-Cas9.
• To investigate the functions of nine previously uncharacterized OsMIRNA genes in rice using CRISPR-Cas12a-mediated knockout.

Main Methods:

• Utilized CRISPR-Cas12a nuclease for targeted genome editing of OsMIR390 and nine other OsMIRNA genes in rice.
• Analyzed deletion sizes at miRNA loci to confirm disruption of pre-miRNA secondary structure.
• Performed transcriptome profiling and phenotypic analysis of resulting rice mutants.

Main Results:

• CRISPR-Cas12a generated significantly larger deletions than Cas9, leading to complete knockout of miRNA genes.
• Achieved up to 100% genome editing efficiency at targeted miRNA loci.
• Identified novel roles for targeted miRNAs in regulating gene expression, rice grain quality, and seed development.

Conclusions:

• CRISPR-Cas12a is a more efficient and robust tool than CRISPR-Cas9 for achieving complete miRNA gene knockouts in plants.
• This study provides a powerful platform for functional genomics of plant miRNAs and crop improvement.