State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, 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, 320 Yueyang Road, Shanghai, China.
CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Genome Biol. 2023 Jan 19;24(1):15. doi: 10.1186/s13059-023-02848-6.
Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export.
We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression.
This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.
在多细胞生物中单细胞中基因转录和 mRNA-蛋白质（mRNP）动力学的理解一直具有挑战性。无酶活性的 CRISPR-Cas13（dCas13）系统已被用于在活细胞中可视化 RNA，而无需遗传操作。我们优化了该系统，以跟踪斑马鱼胚胎中发育表达的 mRNAs，并了解内源性转录动力学和 mRNP 输出的特征。
我们报告说，纯化的 CRISPR-dCas13-荧光蛋白和修饰的指导 RNA 的受精卵微注射允许在斑马鱼胚胎中进行单和双色跟踪，从合子基因组激活（ZGA）到早期分割期，而无需遗传操作。使用这种方法，我们发现等位基因之间的非同步从头转录、等位基因对的同步有丝分裂后再激活以及转录记忆作为可能有助于同步有丝分裂后再激活的外在噪声。我们还揭示了 dCas13 参与的核内 mRNP 的快速运动，具有被束缚的扩散运动，但 mRNP 出口的限速范围很广，可以通过 Alyref 和 Nxf1 的过表达来缩短。
这个优化的基于 dCas13 的工具包能够稳健地进行内源 mRNAs 的时空跟踪，并揭示转录和 mRNP 运动的特征，为多细胞发育生物体中的内源 RNA 可视化提供了强大的工具包。
