CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv. oryzae

Abstract

Background

Genome editing tools are important for functional genomics research and biotechnology applications. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system for gene knockout has emerged as the most effective genome-editing tool. It has previously been reported that, in rice plants, knockdown of the Os8N3 gene resulted in enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), while displaying abnormal pollen development.

Results

The CRISPR/Cas9 system was employed to knockout rice Os8N3, in order to confer enhanced resistance to Xoo. Analysis of the genotypes and edited Os8N3 in T₀, T₁, T₂, and T₃ transgenic rice plants showed that the mutations were transmitted to subsequent generations, and homozygous mutants displayed significantly enhanced resistance to Xoo. Stable transmission of CRISPR/Cas9-mediated Os8N3 gene editing without the transferred DNA (T-DNA) was confirmed by segregation in the T₁ generation. With respect to many investigated agronomic traits including pollen development, there was no significant difference between homozygous mutants and non-transgenic control plants under greenhouse growth conditions.

Conclusion

Data from this study indicate that the CRISPR/Cas9-mediated Os8N3 edition can be successfully employed for non-transgenic crop improvements.