The chaperonin 60 (Cpn60) protein is of great importance to plants due to its involvement in modulating the folding of numerous chloroplast protein polypeptides.

In chloroplasts, Cpn60 is differentiated into two subunit types—Cpn60α and Cpn60β and the rice genome encodes three α and three β plastid chaperonin subunits. However, the functions of Cpn60 family members in rice were poorly understood. In order to investigate the molecular mechanism of OsCpn60β1, we attempted to disrupt the OsCpn60β1 gene by CRISPR/Cas9-mediated targeted mutagenesis in this study. We succeeded in the production of homozygous OsCpn60β1 knockout rice plants. The OsCpn60β1 mutant displayed a striking albino leaf phenotype and was seedling lethal. Electron microscopy observation demonstrated that chloroplasts were severely disrupted in the OsCpn60β1 mutant. In addition, OsCpn60β1 was located in the chloroplast and OsCpn60β1 is constitutively expressed in various tissues particularly in the green tissues. The label-free qualitative proteomics showed that photosynthesis-related pathways and ribosomal pathways were significantly inhibited in OsCpn60β1 mutants. These results indicate that OsCpn60β1 is essential for chloroplast development in rice.





Figure 1. Production of OsCpn60β1 knockout mutants via the CRISPR/Cas9 system. (A) Diagram of CRISPR/Cas9 system for editing OsCpn60β1. (B) Schematic diagram of targets sites in OsCpn60β1. Int. J. Mol. Sci. 2020, 21, 4023 4 of 19 Black boxes show exons, black lines show introns and white boxes show untranslated regions (UTR). (C) Mutation sites of OsCpn60β1 knockout lines. β1-1 mutant has a 38-bp deletion and a 1-bp insertion, which has a 37-bp deletion in total; β1-2 mutant has a 5-bp deletion and a 1-bp insertion, which has a 4-bp deletion in total. (D) Phenotypes of OsCpn60β1 mutants, 7-day-old seedlings were photographed. Scale bar, 1cm.