Rational Improvement of Rice Yield and Cold Tolerance by Editing the Three Genes OsPIN5b, GS3, and OsMYB30 With the CRISPR–Cas9 System

Significant increases in rice yield and stress resistance are constant demands for breeders. However, high yield and high stress resistance are often antagonistic to each other. Here, we report several new rice mutants with high yield and excellent cold tolerance that were generated by simultaneously editing three genes, <i>OsPIN5b</i> (a panicle length gene), <i>GS3</i> (a grain size gene) and <i>OsMYB30</i> (a cold tolerance gene) with the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system. We edited two target sites of each gene with high efficiency: 53% for <i>OsPIN5b</i>-site1, 42% for <i>OsPIN5b</i>-site2, 66% for <i>GS3</i>-site1, 63% for <i>GS3</i>-site2, 63% for <i>OsMYB30</i>-site1, and 58% for <i>OsMYB30</i>-site2. Consequently, the <i>ospin5b</i> mutants, the <i>gs3</i> mutants, and the <i>osmyb30</i> mutants exhibited increased panicle length, enlarged grain size and increased cold tolerance, respectively. Then nine transgenic lines of the <i>ospin5b/gs3</i>, six lines of <i>ospin5b/osmyb30</i> and six lines of <i>gs3/osmyb30</i> were also acquired, and their yield related traits and cold tolerance corresponded to the genes being edited. Additionally, we obtained eight <i>ospin5b/gs3/osmyb30</i> triple mutants by editing all three genes simultaneously. Aside from the <i>ospin5b/gs3/osmyb30-4</i> and <i>ospin5b/gs3/osmyb30-25</i> mutants, the remaining six mutants had off-target events at the putative off-target site of <i>OsMYB30</i>-site1. The results also showed that the T₂ generations of these two mutants exhibited higher yield and better cold tolerance compared with the wild type. Together, these results demonstrated that new and excellent rice varieties with improved yield and abiotic stress resistance can be generated through gene editing techniques and may be applied to rice breeding. Furthermore, our study proved that the comprehensive agronomic traits of rice can be improved with the CRISPR-Cas9 system.