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Nat Commun. 2018 Nov 29;9(1):5065. doi: 10.1038/s41467-018-07498-y.

Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag.

Chen B1,2, Zou W3,4, Xu H5, Liang Y5, Huang B6,7,8.

Author information

1
Department of Cell Biology, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, Hangzhou, 310058, China. baohuichen@zju.edu.cn.
2
Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310003, China. baohuichen@zju.edu.cn.
3
The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, 322000, China.
4
Insititute of Translational Medicine, Zhejiang University, Hangzhou, 310058, China.
5
Department of Cell Biology, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, Hangzhou, 310058, China.
6
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, 94143, CA, USA. bo.huang@ucsf.edu.
7
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, 94143, CA, USA. bo.huang@ucsf.edu.
8
Chan Zuckerberg Biohub, San Francisco, 94158, CA, USA. bo.huang@ucsf.edu.

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.

PMID:
30498221
PMCID:
PMC6265289
DOI:
10.1038/s41467-018-07498-y
[Indexed for MEDLINE]
Free PMC Article

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