Photosynthetic rate at the present atmospheric condition is limited by the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)because of its extremely low catalytic rate(kcat)and poor affinity for CO2(Kc)and specificity for CO2(Sc/o).Rubisco in C4 plants generally shows higher kcat than that in C3 plants.Rubisco consists of eight large subunits and eight small subunits(RbcS).Previously,the chimeric incorporation of sorghum C4-type RbcS significantly increased the kcat of Rubisco in a C3 plant,rice.In this study,we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco.Obtained hybrid Rubisco showed almost C4 plant-like catalytic properties,i.e.,higher kcat,higher Kc,and lower Sc,o.Transgenic lines ex-pressing the hybrid Rubisco accumulated reduced levels of Rubisco,whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO2 condition compared with wild-type rice.High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the βC-βD hairpin in RbcS.We propose that such differences,particularly in the βC-βD hairpin,may impact the flex-ibility of Rubisco catalytic site and change its catalytic properties.