Chapter 21
Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular
Beacon Hybrid Systems
Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger,
and Antony K. Chen
Abstract
The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this
goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been
developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein
9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently
the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with
molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than
conventional approaches employing FP reporters in detecting single telomere loci. In this chapter, we
describe detailed protocols for the synthesis of CRISPR/MB, as well as its applications for imaging single
telomere and centromere loci in live mammalian cells.
Key words CRISPR, Molecular beacons, Chromatin dynamics, Fluorogenic probes
1 Introduction
Over the past several decades, increasing evidence has suggested
that many fundamental cellular processes, including DNA replication, DNA damage repair, and gene expression, are highly regulated by chromatin dynamics [1, 2]. Consequently, much effort has
been devoted to developing methods to enable direct visualization
of single chromatin loci in living cells, with many approaches developed based on gene-editing tools [3, 4]. One such tool is the
clustered regularly interspaced short palindromic repeat (CRISPR)
system [5], which consists of two components: the CRISPRassociated protein 9 (Cas9) DNA nuclease and a chimeric singleguide RNA possessing a Cas9-binding motif and a spacer sequence
complementary to the target DNA sequence of interest [6]. To edit
a specific sequence, the sgRNA recruits Cas9 to form a stable
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_21, © Springer Science+Business Media, LLC, part of Springer Nature 2020
357
Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular
Beacon Hybrid Systems
Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger,
and Antony K. Chen
Abstract
The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this
goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been
developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein
9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently
the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with
molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than
conventional approaches employing FP reporters in detecting single telomere loci. In this chapter, we
describe detailed protocols for the synthesis of CRISPR/MB, as well as its applications for imaging single
telomere and centromere loci in live mammalian cells.
Key words CRISPR, Molecular beacons, Chromatin dynamics, Fluorogenic probes
1 Introduction
Over the past several decades, increasing evidence has suggested
that many fundamental cellular processes, including DNA replication, DNA damage repair, and gene expression, are highly regulated by chromatin dynamics [1, 2]. Consequently, much effort has
been devoted to developing methods to enable direct visualization
of single chromatin loci in living cells, with many approaches developed based on gene-editing tools [3, 4]. One such tool is the
clustered regularly interspaced short palindromic repeat (CRISPR)
system [5], which consists of two components: the CRISPRassociated protein 9 (Cas9) DNA nuclease and a chimeric singleguide RNA possessing a Cas9-binding motif and a spacer sequence
complementary to the target DNA sequence of interest [6]. To edit
a specific sequence, the sgRNA recruits Cas9 to form a stable
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_21, © Springer Science+Business Media, LLC, part of Springer Nature 2020
357