Improving a Quantitative Trait in Rice by Multigene Editing with CRISPR-Cas9

Area of Science:

• Agricultural Science
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas9 is a sequence-specific nuclease (SSN) superior to ZFN and TALEN for multiplex genome editing.
• Multiplex genome editing is crucial for improving crop traits by targeting multiple quantitative trait loci (QTL) simultaneously.
• Applications include large fragment deletion, gene family analysis, exon exchange, and gene regulation.

Purpose of the Study:

• To provide a detailed protocol for constructing a T-DNA binary vector for multiplex CRISPR-Cas9 gene editing.
• To enable simultaneous targeting of three QTL genes for seed trait improvement in rice.

Main Methods:

• Assembly of multiple single-guide RNAs (sgRNAs) into a single binary vector.
• Utilizing Golden Gate assembly for vector construction.
• Developing a T-DNA binary vector for Cas9 and three sgRNAs expression.

Main Results:

• A detailed protocol for constructing a T-DNA binary vector for multiplex CRISPR-Cas9 is presented.
• The method facilitates simultaneous targeting of three QTL genes in rice.

Conclusions:

• Multiplex CRISPR-Cas9, facilitated by efficient vector construction, is a powerful tool for crop trait improvement.
• This protocol supports advanced genetic studies and breeding strategies in rice.