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

Xuelian Zheng1,2, Xu Tang1, Yuechao Wu3,4

  • 1Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Chongqing Key Laboratory of Tree Germplasm Innovation and Utilization, School of Life Sciences, Southwest University, Chongqing, China.

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CRISPR-Cas12a efficiently generates larger deletions than CRISPR-Cas9 for complete knockout of plant microRNA genes, enabling new insights into gene function and crop development.

Keywords:
CRISPR‐Cas12aMicroRNAgenome Editinggermplasm innovationrice

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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.