CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants

Area of Science:

• Plant biotechnology
• Molecular biology
• Genome engineering

Background:

• CRISPR/Cas9 is a powerful genome editing tool with high efficiency and ease of use compared to ZFNs and TALENs.
• Multiplex genome editing allows simultaneous modification of multiple genes, enhancing genetic studies and crop improvement.
• Efficient methods for multiplex genome targeting in plants are crucial for advancing agricultural biotechnology.

Purpose of the Study:

• To develop and present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants.
• To demonstrate the utility of a versatile CRISPR/Cas9 vector system for constructing multiplex genome editing constructs.
• To describe a method for genotyping targeted mutations in transgenic plants.

Main Methods:

• Utilized a CRISPR/Cas9 vector system for multiplex genome targeting in plants.
• Employed Golden Gate ligation for efficient construction of binary constructs with multiple sgRNA expression cassettes.
• Performed genotyping of targeted mutations using direct Sanger sequencing and the DSDecode tool for chromatogram analysis.

Main Results:

• Achieved highly efficient multiplex genome targeting in both monocot and dicot plants.
• Successfully constructed versatile binary constructs with multiple sgRNA expression cassettes in a single cloning step.
• Developed a robust method for genotyping biallelic or heterozygous mutations in transgenic plants.

Conclusions:

• The presented CRISPR/Cas9 procedure enables efficient multiplex genome editing in a wide range of plant species.
• The Golden Gate ligation-based cloning strategy simplifies the construction of multiplex genome editing vectors.
• The described genotyping method facilitates accurate analysis of targeted mutations in edited plants.