SummaryCis‐regulatory element editing can generate quantitative trait variation that mitigates extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed CRISPR/Cas9 approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of rice stomatal density. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild‐type stomatal density. This panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation and intrinsic water use efficiency in steady‐state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought‐responsive stomatal density reprogramming in stomagen and two cis‐regulatory edited alleles was reduced. Collectively our data demonstrate that cis‐regulatory element editing can generate near‐isogenic trait variation that can be leveraged for establishing relationships between anatomy and physiology, providing a basis for optimizing traits across diverse environments.

中文翻译：

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通过顺式调节编辑设计定量气孔性状变异和局部适应潜力


摘要顺式调节元件编辑可以产生数量性状变异，从而减轻与编码序列突变相关的极端表型和有害多效性。在这里，我们应用了一种由生物信息学数据集提供的多重 CRISPR/Cas9 方法，在 OsSTOMAGEN 的启动子中产生基因型变异，OsSTOMAGEN 是水稻气孔密度的正调节因子。工程基因型变异对应于气孔密度的广泛和连续变化，范围为野生型气孔密度的 70% 至 120%。在生理测定中利用这组气孔变异来建立气孔形态变异与稳态和波动光照条件下气孔导度、碳同化和内在水分利用效率之间的离散关系。此外，启动子等位基因受到营养性干旱制度的影响，以测定编辑后的等位基因对干旱发育反应的影响。值得注意的是，气孔和两个顺式调节编辑等位基因中干旱响应气孔密度重编程的能力降低。总的来说，我们的数据表明，顺式调节元件编辑可以产生近同基因性状变异，可用于建立解剖学和生理学之间的关系，为优化不同环境中的性状提供基础。