SummaryRibulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR‐Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1–5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2–5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild‐type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.

中文翻译：

---

通过多重 CRISPR 编辑水稻中的小亚基对 RuBisCO 进行基因工程


摘要核酮糖-1,5-二磷酸羧化酶/加氧酶 （RuBisCO） 是光合碳同化所必需的，因为它催化无机碳转化为有机碳。尽管 RuBisCO 很重要，但它效率低下;它的催化速率低，底物特异性差。提高 RuBisCO 的催化性能是增强植物光合作用的关键途径之一。RbcS 作为 RuBisCO 的基本亚基，影响催化性能，在稳定全酶结构方面起关键作用。然而，对 RbcS 在作物中的功能的认识在很大程度上仍然未知。为此，我们采用 CRISPR-Cas9 技术随机编辑水稻中的 5 个 rbcS 基因 （OsrbcS1-5），生成一系列敲除突变体。水稻光合组织中主要 rbcS 基因 OsrbcS2-5 的突变导致田间条件下生长受抑制、抽穗延迟和产量降低，同时 RuBisCO 含量和活性降低，光合效率显著降低。由多个突变引起的迟缓表型更严重。此外，我们发现这些突变体的叶绿体和淀粉粒较少，芽基部的糖含量较低，导致稻分蘖较少。对一个 rbcs2,3,5 突变系中突变的 RuBisCO 酶的进一步结构分析发现与野生型蛋白没有显着差异，表明 rbcS 的突变不会损害蛋白质组装或结构。我们的研究结果生成了一个具有遗传多样性的突变库，这为揭示水稻中 RuBisCO 的机制提供了宝贵的资源和新颖的见解。 本研究的多重基因工程方法为作物中的 RuBisCO 修饰提供了一种有效可行的策略，进一步促进了光合作用的改进和作物的可持续生产。