High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Agricultural biotechnology

Background:

• CRISPR/Cas9 gene editing efficiency in plants is significantly influenced by promoter selection for Cas9 and sgRNA expression.
• Optimizing promoter elements is crucial for enhancing targeted genome modification in crops.

Purpose of the Study:

• To evaluate the efficiency of a novel CRISPR/Cas9 system in maize utilizing the maize dmc1 gene promoter (DPC) for Cas9 and a U3 promoter for sgRNA.
• To assess the inheritance stability and specificity of mutations generated by the DPC CRISPR/Cas9 system.

Main Methods:

• Targeted genome editing was performed on three distinct loci in the maize genome.
• Regenerated T0 plants were analyzed for editing efficiency, and mutations were tracked through the T1 generation.
• Whole-genome resequencing was employed to detect potential off-target mutations.

Main Results:

• The DPC CRISPR/Cas9 system demonstrated high editing efficiency in maize, with approximately 66% of T0 plants exhibiting homozygous or bi-allelic mutations.
• Mutations were stably transmitted to the T1 generation, and new mutations were observed in gametes and zygotes.
• Whole-genome resequencing confirmed the absence of detectable off-target mutations at predicted loci.

Conclusions:

• The dmc1 promoter-controlled (DPC) CRISPR/Cas9 system is a highly efficient tool for targeted genome editing in maize.
• The findings underscore the importance of promoter optimization for enhancing CRISPR/Cas9 efficacy in plant genome engineering.
• The evolutionary conservation of the dmc1 gene suggests broad applicability of this system across diverse plant species.