complex that can transiently bind to a short DNA sequence known
as the protospacer adjacent motif (PAM) and locally unwind the
DNA duplex [7–9]. Complementation of the sgRNA spacer
sequence with the DNA sequence of the genomic protospacer
stabilizes binding between the complex and the target sequence
[9], allowing Cas9 to introduce double-stranded breaks that subsequently elicit error-prone repair by nonhomologous end joining to
result in generation of new sequences [10–12].
Cas9 modified to eliminate nuclease activity (dCas9) can still
bind specific DNA sequences in a sgRNA-guided fashion. This has
spurred the development of various CRISPR/dCas9-based probes
for live-cell fluorescent labeling of genomic loci, with either the
dCas9 protein or the sgRNA modified to allow tagging by fluorescent proteins (FPs) [13–22], small-molecule dyes [23–26], oligonucleotide probes [27], or quantum dots [28] in a manner that
does not appear to disrupt DNA targeting capacities. In this chapter, we describe the synthesis and applications of one such probe
that was developed in our laboratory, termed the CRISPR/molecular beacon (MB) hybrid system (CRISPR/MB) [27]. Molecular
beacons are a class of stem-loop-forming, fluorogenic oligonucleotide probes that possess a dye and a quencher at the two termini
[29]. In the absence of target, the short arm sequences at the two
termini self-anneal to form a duplex stem, holding the dye and the
quencher in close spatial proximity, causing the MBs to emit a low
fluorescence signal. Hybridization of target sequence to the MB
disrupts the stem, separating the dye from the quencher to restore
MB fluorescence. To image a genomic locus in living cells using
CRISPR/MB (see Fig. 1), dCas9 and an sgRNA engineered to
harbor a unique MB target sequence (sgRNA-MTS) are first
co-expressed. After sufficient time is given to allow dCas9sgRNA-MTS complex to bind a target locus, MBs are delivered
into the cells by microporation. Collective hybridization of the MBs
to the dCas9-sgRNA-MTS complexes tiling across the target locus
can cause the locus to appear as a bright fluorescent spot, indicative
of a single genomic locus, when imaged via fluorescence microscopy. Protocols used for live-cell imaging and tracking of single
telomere and centromere loci are provided.
2 Materials
2.1 Plasmids
1. pSLQ1658-dCas9-EGFP that encodes the nucleasedeactivated Streptococcus pyogenes Cas9 protein (dCas9), fused
to EGFP, is available on Addgene, a nonprofit plasmid repository (Code #51023).
2. pU6-SL2-sgTelo-MTSa and pU6-SL2-sgSat-MTSb (see
Note 1): These two plasmids containing genomic-targeting
sgRNAs harboring two orthogonal MTSs, named MTSa and
358
Xiaotian Wu et al.
as the protospacer adjacent motif (PAM) and locally unwind the
DNA duplex [7–9]. Complementation of the sgRNA spacer
sequence with the DNA sequence of the genomic protospacer
stabilizes binding between the complex and the target sequence
[9], allowing Cas9 to introduce double-stranded breaks that subsequently elicit error-prone repair by nonhomologous end joining to
result in generation of new sequences [10–12].
Cas9 modified to eliminate nuclease activity (dCas9) can still
bind specific DNA sequences in a sgRNA-guided fashion. This has
spurred the development of various CRISPR/dCas9-based probes
for live-cell fluorescent labeling of genomic loci, with either the
dCas9 protein or the sgRNA modified to allow tagging by fluorescent proteins (FPs) [13–22], small-molecule dyes [23–26], oligonucleotide probes [27], or quantum dots [28] in a manner that
does not appear to disrupt DNA targeting capacities. In this chapter, we describe the synthesis and applications of one such probe
that was developed in our laboratory, termed the CRISPR/molecular beacon (MB) hybrid system (CRISPR/MB) [27]. Molecular
beacons are a class of stem-loop-forming, fluorogenic oligonucleotide probes that possess a dye and a quencher at the two termini
[29]. In the absence of target, the short arm sequences at the two
termini self-anneal to form a duplex stem, holding the dye and the
quencher in close spatial proximity, causing the MBs to emit a low
fluorescence signal. Hybridization of target sequence to the MB
disrupts the stem, separating the dye from the quencher to restore
MB fluorescence. To image a genomic locus in living cells using
CRISPR/MB (see Fig. 1), dCas9 and an sgRNA engineered to
harbor a unique MB target sequence (sgRNA-MTS) are first
co-expressed. After sufficient time is given to allow dCas9sgRNA-MTS complex to bind a target locus, MBs are delivered
into the cells by microporation. Collective hybridization of the MBs
to the dCas9-sgRNA-MTS complexes tiling across the target locus
can cause the locus to appear as a bright fluorescent spot, indicative
of a single genomic locus, when imaged via fluorescence microscopy. Protocols used for live-cell imaging and tracking of single
telomere and centromere loci are provided.
2 Materials
2.1 Plasmids
1. pSLQ1658-dCas9-EGFP that encodes the nucleasedeactivated Streptococcus pyogenes Cas9 protein (dCas9), fused
to EGFP, is available on Addgene, a nonprofit plasmid repository (Code #51023).
2. pU6-SL2-sgTelo-MTSa and pU6-SL2-sgSat-MTSb (see
Note 1): These two plasmids containing genomic-targeting
sgRNAs harboring two orthogonal MTSs, named MTSa and
358
Xiaotian Wu et al.