State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China.
Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
Sci China Life Sci. 2024 Nov;67(11):2297-2306. doi: 10.1007/s11427-023-2572-6. Epub 2024 Aug 2.
The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
不断增加的 RNA 种类，如 mRNAs、lncRNAs 和 circRNAs，在发育过程和各种病理生理条件中发挥关键作用。然而，由于缺乏持久有效的直接影响 RNA 水平的策略，我们对活生物体中 RNA 功能的理解仍然有限。在这项研究中，我们将 CRISPR-RfxCas13d 系统与精子样干细胞介导的半克隆技术相结合，从而能够抑制不同 RNA 种类的表达。该方法用于干扰三种 RNA 分子的表达：Sfmbt2 mRNA、Fendrr lncRNA 和 circMan1a2(2,3,4,5,6)。结果证实了这些 RNA 在胚胎发育中的关键作用，因为它们的缺失导致了明显的表型，包括胚胎致死、胚胎发育迟缓以及胚胎吸收。总之，我们的方法为在活生物体中沉默特定的 RNA 靶标提供了一种强大的工具包，而无需引入遗传改变。
