Key message

We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color.

The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar ‘Madness Midnight’ has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T₀ plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer’s field.

中文翻译：

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通过 CRISPR-Cas9 核糖核蛋白同时靶向矮牵牛原生质体中的重复基因以修饰花色。

 关键信息

我们通过Cas9-核糖核蛋白递送获得了两个F3H基因均发生突变的矮牵牛完整突变系，其呈现淡紫粉色的花色。

CRISPR-Cas系统现在正在彻底改变农业，它允许研究人员随意在植物中产生各种所需的突变。特别是，通过 Cas9-核糖核蛋白 (RNP) 传递进行的无 DNA 基因组编辑在植物中具有许多优势；它不需要密码子优化或特定启动子即可在植物细胞中表达；此外，它可以绕过一些国家的转基因法规。在这里，我们在矮牵牛中进行了位点特异性诱变，以改变花的颜色。我们确定商业矮牵牛品种“Madness Midnight”具有两个F3H编码基因，并设计了一种同时靶向两个F3H基因的引导 RNA。在从Cas9-RNP转染的原生质体再生的67个T ₀植物中，我们获得了7个含有F3HA或F3HB基因突变的突变株系和1个在两个F3H基因中都含有突变但没有任何选择标记的完整突变株系。值得注意的是，只有f3ha f3hb表现出明显修饰的淡紫粉色花朵颜色 (RHS 69D)，而其他植物，包括单拷贝基因敲除植物，则表现出与野生植物相似的紫紫色 (RHS 93A) 花朵。类型矮牵牛。据我们所知，我们首次展示了利用无DNA CRISPR方法进行观赏作物工程的先例，这将大大加速从实验室到农民田间的转变。