Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 30498221.
- Also identified by DOI 10.1038/s41467-018-07498-y and PMC identifier 6265289.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
The lack of efficient tools to image non-repetitive genes in living cells has limited our ability to explore the functional impact of the spatiotemporal dynamics of such genes. Here, we addressed this issue by developing a CRISPR-Tag system using one to four highly active sgRNAs to specifically label protein-coding genes with a high signal-to-noise ratio for visualization by wide-field fluorescence microscopy. Our approach involves assembling a CRISPR-Tag within the intron region of a fluorescent protein and then integrating this cassette to N- or C-terminus of a specific gene, which enables simultaneous real-time imaging of protein and DNA of human protein-coding genes, such as HIST2H2BE, LMNA and HSPA8 in living cells. This CRISPR-Tag system, with a minimal size of ~250 bp DNA tag, represents an easily and broadly applicable technique to study the spatiotemporal organization of genomic elements in living cells.
Medical subject headings
- Clustered Regularly Interspaced Short Palindromic Repeats
- Luminescent Proteins
- Microscopy, Fluorescence