Grenoble Alpes University-CNRS-INRAE-CEA, Plant and Cell Physiology Lab (LPCV), IRIG-DBSCI, Grenoble, France.
Methods Mol Biol. 2025;2873:305-332. doi: 10.1007/978-1-0716-4228-3_17.
Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.
开发针对染色质上特定区域的基因座/转录可视化或转录/表观遗传标记编辑的局部方法是功能遗传学和表观遗传学的关键挑战。基于成簇规律间隔短回文重复序列 (CRISPR) 及其相关内切酶 (Cas) 的系统通过所谓的向导 RNA 进行 DNA 序列特异性识别,从而为精确的染色质可视化或编辑提供了高度的灵活性和模块化。在这里,我们概述了为活体成像中染色质基因座可视化或有效修饰基因表达而开发的基于 CRISPR/Cas 的工具。这些工具利用效应模块,将激活剂、抑制剂和表观遗传修饰剂与失活的 Cas 蛋白 (dCas) 结合。我们展示了它们在植物中的应用如何在可视化或操纵与农艺性状(如开花时间和对干旱或高温的响应)相关的基因座表达方面取得了进展。我们还讨论了 dCas 相关技术的局限性和未来改进,例如更紧凑和组合系统、时空靶向以精细调节基因表达,以及染色质动力学的实时可视化。