Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

Area of Science:

• Plant genetics
• Molecular biology
• Crop science

Background:

• CRISPR/Cas9 is a powerful tool for crop genetic breeding.
• Ubiquitous CRISPR/Cas9 expression leads to numerous somatic mutations, hindering heritable mutation detection.
• Targeting gene editing to specific tissues can potentially improve efficiency.

Purpose of the Study:

• To develop and evaluate a callus-specific CRISPR/Cas9 (CSC) system in maize.
• To compare the CSC system with a ubiquitously expressed CRISPR/Cas9 (UC) system.
• To assess the impact of somatic mutations on heritable mutation detection and stability.

Main Methods:

• Constructed a CSC system using maize callus-specific promoters (pZmCTA1, pZmPLTP) to drive Cas9 expression.
• Targeted the bZIP transcription factor Opaque2 (O2) gene for editing.
• Utilized high-throughput tracking of mutations (Hi-TOM) for editing efficiency analysis.
• Generated transgenic plants and analyzed mutations in T0 and T1 generations.

Main Results:

• CSC systems generated more target gene mutations in calli compared to the UC system.
• CSC systems produced fewer target gene mutations in T0 seedlings but reduced the influence of somatic mutations.
• Nearly 100% of T1 mutations from CSC systems were heritable from T0 plants, versus 6.3-16.7% for the UC system.

Conclusions:

• The CSC system significantly reduces somatic mutations and enhances the production of stable, heritable mutations in maize.
• This system offers a promising approach for precise genetic breeding across various crops.
• Improved heritable mutation detection facilitates the development of crops with desired traits.