生物
斑马鱼
母子转换
清脆的
抄写(语言学)
遗传学
多细胞生物
细胞生物学
核糖核酸
基因
合子
胚胎发生
语言学
哲学
作者
Youkui Huang,Bao-Qing Gao,Quan Meng,Liang-Zhong Yang,Xu-Kai Ma,Hao Wu,Yu-Hang Pan,Li Yang,Dong Li,Ling-Ling Chen
标识
DOI:10.1186/s13059-023-02848-6
摘要
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.
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