CRISPR/Cas9定点编辑水稻LOC_Os05g31750基因位点及靶修饰位点遗传稳定性研究

Study on Hereditary Stability of the LOC_Os05g31750 Locus via CRISPR/Cas9-mediated Genome Editing in Rice

为明确CRISPR/Cas9系统中Cas9蛋白的持续剪切是否会导致原已突变的靶位点在水稻代际之间传递时再次产生新的突变,本研究以水稻粳稻品种台北309为转基因受体材料,通过CRISPR/Cas9技术定点编辑了水稻基因位点LOC_Os05g31750,并获得了6株靶位点敲除成功的T_0转基因突变体植株,经三代种植收获后,进一步对携带Cas9基因的T_1和T_2群体进行靶修饰位点PCR扩增测序检测。结果表明,来源于2个T_0纯合双等位突变体T_0-8和T_0-12的所有测试后代群体的靶修饰位点序列都与对应的T_0突变体保持一致,进一步证明CRISPR/Cas9基因编辑水稻的靶修饰位点在代际之间可以稳定遗传。本研究结果为利用CRISPR/Cas9基因编辑突变体的遗传后代进行基因功能丧失后的表型分析提供了参考依据。

In order to illuminate whether new mutations will take place in target modification site when the mutated locus passed from rice with continuous shearing of Cas9 protein after multiple generations,in this study,the japonica rice variety Taipei309 was used as the transgene receptor,and the LOC_Os05 g31750 locus was knocked out using CRISPR/Cas9-mediated genome editing. Six T0 mutated plantlets were obtained successfully. After the harvest of several generations,the target modification sites from T1 and T2 progeny population carrying Cas9 gene were amplified and sequenced. The results showed that the target modification site in progeny population from two homozygous biallelic mutants T0-8 and T0-12 were consistent with the corresponding T0 generation mutant. It was confirmed that the target modification sites via CRISPR/Cas9 gene editing could be reliably inherited between multiple generations from rice. The results provide experimental evidence for verifying the progenis of mutant from CRISPR/Cas9 genome editing could be used for analysis of phenotypes after gene function loss.