Engineering TGMS in rice through CRISPR/Cas9-mediated genome editing

The thermosensitive genic male sterility (TGMS) system is widely used for hybrid rice cultivation. Male sterile lines are sensitive to temperature and this trait is controlled by the TMS5 gene, encoding a nuclear ribonuclease Z (RNase Z^S1) enzyme, which is involved in the degradation of ubiquitin fusion ribosomal protein L40 (Ub_L40) mRNAs. Mutation in the TMS5 gene will lead to loss of function of RNase Z^S1, resulting in accumulation of high levels of Ub_L40 mRNAs, thereby causing male sterility. We developed tms5 mutants through targeted mutagenesis of the TMS5 gene via CRISPR/Cas9 technology in three rice genetic backgrounds, viz., CO51, ADT39, and ASD16. The tms5 mutants exhibited pollen sterility percentages ranging from 2.05 to 91.1% and seed setting rates between 1.14 and 97.36% under restrictive temperature. This indicates that the mutants possess traces of fertile pollen due to incomplete suppression of RNase Z^S1, resulting in partial fertility even under restrictive temperature. Hence, we conclude that the complete suppression of RNase Z^S1 activity in tms5 mutants is necessary for using them as female parents in hybrid rice breeding programs.