Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
University of the Chinese Academy of Sciences, Beijing 100049, China.
Cell Rep Methods. 2023 Jan 17;3(1):100389. doi: 10.1016/j.crmeth.2022.100389. eCollection 2023 Jan 23.
Gene targeting (GT) is a powerful tool for modifying endogenous genomic sequences of interest, such as sequence replacement and gene knockin. Although the efficiency of GT is extremely low in higher plants, engineered sequence-specific nucleases (SSNs)-mediated double-strand breaks (DSBs) can improve GT frequency. We recently reported a CRISPR-Cas9-mediated approach for heritable GT in , called the "sequential transformation" strategy. For efficient establishment of GT via the sequential transformation method, strong Cas9 activity and robust DSBs are required in the plant cells being infected with carrying sgRNA and donor DNA. Accordingly, we generated two independent parental lines with maize promoter-driven Cas9 and established sequential transformation-mediated GT in the Japonica rice cultivar Nipponbare. We achieved precise knockin into the endogenous and loci. We believe that our GT technology could be widely utilized in rice research and breeding applications.
基因打靶（GT）是一种强大的工具，可用于修饰感兴趣的内源性基因组序列，例如序列替换和基因敲入。尽管 GT 在高等植物中的效率极低，但工程序列特异性核酸酶（SSNs）介导的双链断裂（DSBs）可以提高 GT 频率。我们最近报道了一种在 中进行可遗传 GT 的 CRISPR-Cas9 介导的方法，称为“顺序转化”策略。为了通过顺序转化方法有效地建立 GT，携带 sgRNA 和供体 DNA 的被感染的植物细胞需要具有强的 Cas9 活性和稳健的 DSBs。因此，我们生成了两个具有独立亲本系的玉米 启动子驱动的 Cas9，并在粳稻品种 Nipponbare 中建立了顺序转化介导的 GT。我们实现了内源 和 基因座的精确敲入。我们相信我们的 GT 技术可以在水稻研究和育种应用中得到广泛应用。
