Abstract
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.
Highlights
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation
To test whether the extended sgRNAs form a functional complex with dCas[9] while binding to MS2 coat protein (MCP)–YFP, we co-transfected human cervical cancer (HeLa) cells with dCas9-mCherry, MCP–YFP, and a single sgRNA 14 Â -MS2 targeting an 84-repeat sequence located within the MUC4 gene (Fig. 1b)
Chromatin immunoprecipitation and quantitative PCR (ChIP–qPCR) assays verified that both dCas[9] and MCP were substantially enriched at the MUC4 site (Fig. 1c,d)
Summary
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. We design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. To study chromatin dynamics in living cells, zinc fingers (ZNF)[5] and transcription activator–like effector (TALE) proteins[6] have been engineered to target repetitive genomic regions, such as centromeres and telomeres, and track the spatiotemporal dynamics of these regions in live cells Despite these advances, these approaches are difficult to implement for imaging non-repetitive genomic loci, since they require constructing a large array of TALEs/ZNFs proteins targeting the same locus. Extended sgRNAs enabled us to track the targeted loci over the course of the entire cell cycle, and to distinguish the nuclear positions of various transcriptionally active and inactive sites in the genome of live cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
