Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T₂ to T₄ generations, of which the selection of T₁ and T₂ was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T₂ plants. All indel mutants passed stably to T₃ or T₄ without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.

尽管簇状规则间隔的短回文重复序列（CRISPR）/ CRISPR相关9（Cas9）系统已被广泛用于模型植物的基础研究，但其在作物应用育种中的应用却面临着强大的监管障碍，这主要是由于人们对其了解不足该系统的真实输出，尤其是在更高世代中。在这项研究中，从任何以前的研究不同，我们调查了详细的分子特征和CRISPR / Cas9生成的生产性能SD1（半矮化1）在T突变体₂至T ₄代，其中T的选择₁和Ť ₂仅通过视觉上的矮矮植物表型来完成。我们的数据不仅揭示了带有较小或较大插入缺失的靶上和脱靶突变，而且还揭示了T ₂植物中的外源元件。所有indel突变体都稳定地传递给T ₃或T _4，而没有其他修饰，而与Cas9的存在无关，而某些品系显示出Cas9或某些外源元件的意外遗传模式。此外，各种SD1的效果水稻高度和产量的等位基因取决于遗传背景。综上所述，我们的数据表明，CRISPR / Cas9系统可有效产生纯合突变体以进行功能分析，但它可能不如水稻预期的那么精确，并且必须先进行子代的早期准确分子鉴定和筛选CRISPR / Cas9系统从实验室到现场的过渡。