Abstract
The tagging of genomic loci in living cells provides 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/dCas9-SunTag 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.
Highlights
Fluorescent labeling of genes provides visual information for cell biology research
It has been reported that in HeLa cells, tandem GFP proteins with sizes ranging from 90 to 110 kD were allowed to diffuse through the nuclear pore, and a 5-copy GFP fusion with a molecular weight of approximately 135 kD was mainly distributed in the cytoplasm (Wang and Brattain, 2007)
The molecular size of the scFv-superfolder GFP (sfGFP)-nEGFP labeling modules without an nuclear location sequence (NLS) ranged from 62.7 kD to 145 kD, which was within the critical range of the nuclear pore size
Summary
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/ dCas (clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling (Chen et al, 2013). FISH can obtain the relative spatial position information for several gene loci (Boyle et al, 2001; Roix et al, 2003). It can only be used in fixed cells, mainly because the denaturation of DNA is required for the accessibility of the probe, and it cannot provide dynamic information. This technique has been used to label highly repetitive sequences such as telomeres and centromeres
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