Altering a trait by CRISPR-Cas9-targeted mutagenesis offers great advantages in identifying gene function and crop improvement. In the present study, three genes (OsF3′H, OsDFR and OsLDOX) in the anthocyanin biosynthesis pathway were successfully edited on the Heugseonchal or Sinmyungheugchal variety using the CRISPR/Cas9 system. As a result, the ratio of the edited plants in the transformed early generation was 56.7%. These edited mutant lines were observed with the changes of seed color and anthocyanin content. All mutations were stably inherited to the T₂ progeny. In addition, we could select edited homozygous mutant lines lacking the T-DNA already in the first offspring generation. Also the insertion of vector backbone sequences in f3′h-9, dfr-4 and ldox-16 lines was not detected in the whole genome resequencing. These results demonstrated that the CRISPR/Cas9 system can induce clearly gene-specific mutations with a high efficiency in rice and null plants selected from these mutants cannot be distinguished from non-GMO plants even under strict GMO regulation.

中文翻译：

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CRISPR / Cas9靶向诱变的F3'H，DFR和LDOX，与黑米（Oryza sativa L.）花色苷生物合成相关的基因

通过以CRISPR-Cas9为靶点的诱变来改变性状在鉴定基因功能和作物改良方面具有巨大优势。在本研究中，使用CRISPR / Cas9系统成功地在Heugseonchal或Sinmyungheugchal变种上成功编辑了花色苷生物合成途径中的三个基因（OsF3'H，OsDFR和OsLDOX）。结果，在转化的早代中编辑植物的比例为56.7％。随着种子颜色和花青素含量的变化，观察到这些编辑的突变体系。所有突变均稳定地遗传给T ₂后代。此外，我们可以选择在第一代后代中已经缺少T-DNA的编辑纯合突变株。同样在f3'h-9中插入载体主链序列，在整个基因组重测序中未检测到dfr -4和ldox-16品系。这些结果表明，CRISPR / Cas9系统可以在水稻中高效诱导明显的基因特异性突变，即使在严格的GMO调控下，从这些突变体中选出的无效植物也无法与非GMO植物区分开。