Illuminating single genomic loci in live cells by reducing nuclear background fluorescence
- PDF / 5,445,935 Bytes
- 11 Pages / 595.276 x 793.701 pts Page_size
- 100 Downloads / 191 Views
luminating single genomic loci in live cells by reducing nuclear background fluorescence 1,2
2
2,3
2
1†*
Song Lu , Dianbing Wang , Yu Hou , Dongge Guo , Yulin Deng
& Xian-En Zhang
2,3†*
1
2
School of Life Sciences, Beijing Institute of Technology, Beijing 100081, China; National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 3 University of Chinese Academy of Sciences, Beijing 100049, China Received June 30, 2020; accepted August 10, 2020; published online October 26, 2020
The tagging of genomic loci in living cells provide visual evidence for the study of genomic spatial organization and gene interaction. CRISPR/dCas9 (clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci. However, the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout. This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9SunTag labeling system consisting of dCas9-SunTag-NLS (target module) and scFv-sfGFP-NLS (signal module). We removed the nuclear location sequence (NLS) of the signal module and inserted two copies of EGFP into the signal module. The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm (N/C ratio) was decreased by 71%, and the ratio of the signal to the background (S/B ratio) was increased by 1.6 times. The system can stably label randomly selected genomic loci with as few as 9 repeat sequences. CRISPR/dCas9 labeling, background reduction, low-repetitive loci, live cell imaging Citation:
Lu, S., Wang, D., Hou, Y., Guo, D., Deng, Y., and Zhang, X.E. (2020). Illuminating single genomic loci in live cells by reducing nuclear background fluorescence. Sci China Life Sci 63, https://doi.org/10.1007/s11427-020-1794-2
INTRODUCTION Fluorescent labeling of genes provides visual information for cell biology research. Many methods have been developed for these purposes, such as fluorescence in situ hybridization (FISH) (Narayanswami and Hamkalo, 1990; Pinkel et al., 1986), transcription activator-like effector (TALE)-mediated genome visualization (Miyanari et al., 2013), and CRISPR/ dCas9 (clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling (Chen et al., 2013). FISH can obtain the relative spatial position information for sev-
†Contributed equally to this work *Corresponding authors (Yulin Deng, email: [email protected]; Xian-En Zhang, email: [email protected])
eral gene loci (Boyle et al., 2001; Roix et al., 2003). However, it can only be used in fixed cells, mainly because the denaturation of DNA is required for the accessibility of the probe, and thus it cannot provide dynamic information. The TALE protein contains a central repeat domain, which mediates DNA recognition, and each rep
Data Loading...
