CRISPR/Cas9-Mediated Multiplex Genome Editing of the

Area of Science:

• Plant biotechnology
• Molecular genetics
• Crop improvement

Background:

• The clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 system is a key tool for plant genome editing.
• WRKY transcription factors play roles in plant defense pathways, including responses to pathogens like Sclerotinia sclerotiorum.
• BnWRKY11 and BnWRKY70 genes in Brassica napus (rapeseed) are implicated in pathogen resistance.

Purpose of the Study:

• To utilize the CRISPR/Cas9 system for targeted genome editing of BnWRKY11 and BnWRKY70 genes in Brassica napus.
• To investigate the role of BnWRKY70 in Sclerotinia sclerotiorum resistance in Brassica napus.
• To assess the potential of CRISPR/Cas9 for generating disease-resistant rapeseed germplasm.

Main Methods:

• Designing Cas9/sgRNA constructs to target BnWRKY11 and BnWRKY70 genes.
• Generating transgenic Brassica napus plants (T₀ generation) with targeted mutations.
• Analyzing mutations in T₀ and subsequent T₁ generations for efficiency and heritability.
• Phenotypic evaluation of mutant lines for resistance to Sclerotinia sclerotiorum.

Main Results:

• Successfully generated independent transformants with mutations in BnWRKY11 (54.5% mutation rate) and BnWRKY70 (50% mutation rate).
• Obtained plants with mutations in multiple copies of the target genes, with high efficiency observed in the T₁ generation.
• BnWRKY70 mutants displayed enhanced resistance to Sclerotinia sclerotiorum, whereas BnWRKY11 mutants showed no significant difference.
• Overexpression of BnWRKY70 led to increased sensitivity to Sclerotinia, indicating a negative regulatory role.

Conclusions:

• BnWRKY70 acts as a negative regulator of Sclerotinia resistance in Brassica napus.
• The CRISPR/Cas9 system is effective for generating targeted mutations in Brassica napus for crop improvement.
• CRISPR/Cas9-mediated editing of BnWRKY70 offers a promising strategy for developing rapeseed with enhanced Sclerotinia resistance.