A Comparison of Two Versions of the CRISPR-Sirius System for the Live-Cell Visualization of the Borders of Topologically Associating Domains
Author:
Viushkov Vladimir S.1ORCID, Lomov Nikolai A.1ORCID, Rubtsov Mikhail A.12ORCID
Affiliation:
1. Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia 2. Department of Biochemistry, Center for Industrial Technologies and Entrepreneurship, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow 119435, Russia
Abstract
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.
Funder
Russian Science Foundation
Reference56 articles.
1. Huang, S., Dai, R., Zhang, Z., Zhang, H., Zhang, M., Li, Z., Zhao, K., Xiong, W., Cheng, S., and Wang, B. (2023). CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis. Int. J. Mol. Sci., 24. 2. Live-cell imaging of chromatin contacts opens a new window into chromatin dynamics;Grosveld;Epigenet. Chromatin,2023 3. Lu, S., Hou, Y., Zhang, X.E., and Gao, Y. (2023). Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques. Front. Cell Dev. Biol., 11. 4. Visualizing the Nucleome Using the CRISPR-Cas9 System: From in vitro to in vivo;Maloshenok;Biochemistry,2023 5. Thuma, J., Chung, Y.C., and Tu, L.C. (2023). Advances and challenges in CRISPR-based real-time imaging of dynamic genome organization. Front. Mol. Biosci., 10.
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