Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia.
Department of Biochemistry, Center for Industrial Technologies and Entrepreneurship, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow 119435, Russia.
Cells. 2024 Aug 27;13(17):1440. doi: 10.3390/cells13171440.
In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method.
近年来,通过催化失活 Cas9(dCas9)在活细胞中对染色质位进行成像的各种技术已经出现。这些技术有助于更深入地了解染色质动力学背后的机制,并提供了以前通过应用于固定细胞的 FISH 无法获得的有价值的动力学数据。然而,这些技术相对复杂,因为它们涉及表达几种嵌合蛋白以及靶向可视化位的 sgRNA,这一过程涉及许多技术细节。因此,对特定靶位进行可视化的效率可能相当低。在这项研究中,我们直接比较了两种基于使用包含八个 MS2 或 PP7 茎环的 sgRNA 以及融合到荧光蛋白的 MCP 或 PCP 的以前发表的 CRISPR-Sirius 方法的版本。我们通过比较两种方法(瞬时转染和慢病毒转导)以及两种 CRISPR-Sirius 版本(带有 PCP 和 MCP)在递送 sgRNA 基因方面,评估了几个独特基因组位的可视化效率。可视化效率因位而异,并非所有位都能可视化。然而,与 PCP-sfGFP 相比,MCP-sfGFP 在信号细胞数量方面提供了更有效的可视化。我们还表明,对于两种 CRISPR-Sirius 系统,慢病毒转导在定位成像方面比瞬时转染更有效。我们研究中的大多数靶位都位于拓扑关联域的边界处,我们定义了一组 TAD 边界,这些边界可以使用 CRISPR-Sirius 系统的 MCP-sfGFP 版本有效地可视化。总的来说,我们的研究验证了 CRISPR-Sirius 技术在活细胞可视化中的应用,并强调了在使用该方法时应考虑的各种技术细节。