Development of Fragrant Japonica Rice by CRISPR/Cas9-targeted Editing on Badh2

  Objective  The CRISPR/Cas9 technology was applied to edit the Badh2 in rice to enrich the fragrance of current japonica varieties for marketing enhancement.

  Method  A CRISPR/Cas9-BADH vector was constructed to transform 3 elite japonica rice, Longdao 18, Longdao 24, and Xiushui 134. The mutant badh2 was identified by sequencing, and potential off-target mutations examined. Contents of the aromatic 2-acetyl-1-pyrroline (2AP) in the mutant rice cultivars was determined by gas chromatography-mass spectrometry.

  Result  Of the 30 T₀ transgenic plants, 24 were found to contain the target mutant badh2, of which, 53.3% were heterozygous, 16.67% homozygous, and 10% bi-allelic. Seven homozygous mutation genotypes were obtained in the T₁ non-transgenic mutant plants. There was no off-target mutation detected at all 5 potential sites indicating that a high specificity of the designed sgRNA for the predicted site. Interferences by the varied genome backgrounds of the rice varieties might have caused the significantly varied amounts of the aromatic 2AP detected in all badh2 frame-shift mutant rice.

  Conclusion  The CRISPR/Cas9 technology could effectively induce desired Badh2 mutation in rice. Improvement on the fragrance for 3 japonica rice could lead to wide applications in cultivating new varieties with added commercial value. More interestingly, the significant variations on 2AP content among the mutant rice as discovered in this study would help further the understanding on the genetics associated with aroma rice breeding.