Abstract
Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.
Highlights
Accumulating evidence has revealed that the structure of the mammalian genome is organized and regulated in space and time[1,2,3,4], accompanying development, cell proliferation, and cell differentiation[5,6], while disorder of nuclear reorganization can lead to some human diseases[7]
For the bimolecular fluorescence complementation (BiFC), we used the C-terminal fragment (VC155) and the N-terminal fragment (VN173)[28] split from mVenus fluorescent protein. We first fused these fragments to the C-terminus of a pair of transcription activator-like effector (TALE) modules designed such that they bound head-to-head to the target sites on different strands of the double-stranded DNA
The architecture and spatiotemporal organization of chromosomes is essential in biological processes and it would be of great help to have tools for the visualization of specific genomic loci in living cells
Summary
Accumulating evidence has revealed that the structure of the mammalian genome is organized and regulated in space and time[1,2,3,4], accompanying development, cell proliferation, and cell differentiation[5,6], while disorder of nuclear reorganization can lead to some human diseases[7]. TALEs were originally discovered from the plant pathogenic bacteria Xanthomonas and they can bind to target DNA sequences through ~34-aa repeats Each such repeat can recognize a specific single base pair through two adjacent amino acids, called repeat-variable diresidues (RVDs)[10,11]. The most widely used CRISPR system derived from Streptococcus pyogenes is the type II CRISPR/Cas[9] system and it uses a small guide (sg) RNA to help a Cas[9] protein to recognize DNA sequences[12,13] Both the transcription activator-like effector (TALE)[14,15] and the clustered regulatory interspaced short palindromic repeats (CRISPR/Cas) systems[16] have been shown to be promising tools for live-cell genomic fluorescent labeling. With BiFC-TALE, the SBR is >8-fold higher than in the TALE method with full-length FP
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