Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.

中文翻译：

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使用植物中预先组装的CRISPR / Cas9核糖核蛋白进行无DNA基因组编辑。

植物传统性状发育的过程取决于自发突变或人工随机诱变产生的遗传变异。可交亲缘中所需性状的可用性有限，或者无法通过随机诱变生成所需表型，导致开发出真正跨物种且精确的创新育种方法。为此，我们设计了精确的基因组工程的新方法，其特征是使用预先组装的CRISPR / Cas9核糖核蛋白（RNP）复合物，而不是使用核酸和农杆菌。我们发现我们的方法成功地工程了植物基因组，而在基因组中没有留下任何外来的DNA足迹。为了促进将RNP引入植物核中，我们在去除转染屏障，细胞壁后首先获得了原生质体。从已经用RNP工程化的原生质体单细胞再生了整个植物。在改良原生质体再生技术的方法之前，尤其是在农作物中，我们的方法应有助于在短时间内以安全，准确的方式在农作物中开发新特性。