PDF(Pubmed)
Abstract：
Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.
摘要：
基因打靶（GT）允许精确操作基因组序列，如敲入和序列替换，但是种子植物中的GT仍然是一项具有挑战性的任务。已知工程化的序列特异性核酸酶(SSN)在生物体中通过同源定向修复(HDR)促进GT。这里,我们证明了Cas12a和耐温Cas12a变体（ttCas12a）可以通过顺序转化策略在拟南芥（拟南芥）的两个基因座上有效地建立精确和可遗传的GT。因此,ttCas12a显示出比未修饰的Cas12a更高的GT效率。此外,还研究了通过顺序转化策略对GT的转录和翻译增强子的效率。这些增强剂及其组合有望在顺序转化策略中显示GT效率的增加，类似于以前的一体化战略报告，但只观察到最大两倍的增加。这些结果表明，靶位点的双链断裂（DSB）频率是决定植物中GT遗传效率的最重要因素之一。另一方面,更高的DSB频率并不总是导致更高的GT效率，这表明GT通过HDR需要一些额外的因素。因此,不再期望DSB的增加能提高GT效率，未来需要建立新的战略。这项研究为植物中精确和可遗传的GT技术开辟了广泛的应用。
