Methods and compositions for labeling a single-stranded target nucleic acid

A method of detecting a single stranded target nucleic acid in a sample, wherein the target nucleic acid comprises a first target site comprising a first sequence of nucleotides and a second target site comprising a second sequence of nucleotides, wherein the second target site is positioned on the target nucleic acid 5′ of the first target site, the method comprising:

• (I) contacting the sample with:
• (a) a quenched PAMmer, wherein the quenched PAMmer is a single stranded oligonucleotide comprising:
• (a.i) a protospacer adjacent motif (PAM) sequence,
• (a.ii) a detectable label,
• (a.iii) a quencher moiety that quenches the detectable label, and
• (a.iv) at least one of:
• a specificity segment, positioned 5′ of the PAM sequence, that hybridizes with nucleotides of the first target site, and
• an orientation segment, positioned 3′ of the PAM sequence, that hybridizes with nucleotides of the second target site;
• (b) a Cas9 protein;
• and
• (c) a Cas9 guide RNA that forms a complex with the Cas9 protein and hybridizes with the first target site,
• whereby the Cas9 protein cleaves the quenched PAMmer at a cleavage site positioned between the detectable label and the quencher moiety to produce:
• (i) a first cleavage product that is hybridized with the target nucleic acid and comprises the detectable label;
• and (ii) a second cleavage product that is not hybridized with the target nucleic acid and comprises the quencher moiety;
• and
• (II) measuring a signal from the detectable label.

A composition comprising:

• (i) a quenched PAMmer, wherein the quenched PAMmer is a single stranded oligonucleotide comprising:
• (a) a protospacer adjacent motif (PAM) sequence,
• (b) a detectable label,
• (c) a quencher moiety that quenches the detectable label, and
• (d) at least one of:
• a specificity segment, positioned 5′ of the PAM sequence, that hybridizes with nucleotides of a first target site of a target nucleic acid, and
• an orientation segment, positioned 3′ of the PAM sequence, that hybridizes with nucleotides of a second target site of the target nucleic acid;
• and
• (ii) a Cas9 guide RNA comprising a nucleotide sequence that hybridizes with the first target site of the target nucleic acid.

A kit comprising:

• (i) a quenched PAMmer, wherein the quenched PAMmer is a single stranded oligonucleotide comprising:
• (a) a protospacer adjacent motif (PAM) sequence,
• (b) a detectable label,
• (c) a quencher moiety that quenches the detectable label, and
• (d) at least one of:
• a specificity segment, positioned 5′ of the PAM sequence, that hybridizes with nucleotides of a first target site of a target nucleic acid, and
• an orientation segment, positioned 3′ of the PAM sequence, that hybridizes with nucleotides of a second target site of the target nucleic acid;
• and
• (ii) a Cas9 guide RNA comprising a nucleotide sequence that hybridizes with the first target site of the target nucleic acid;
• wherein (i) and (ii) are present in the same or separate containers.

A library comprising:

• two or more labeling nucleic acid pairs, wherein each labeling nucleic acid pair comprises:
• (i) a quenched PAMmer, wherein the quenched PAMmer is a single stranded oligonucleotide comprising:
• (a) a protospacer adjacent motif (PAM) sequence,
• (b) a detectable label,
• (c) a quencher moiety that quenches the detectable label, and
• (d) at least one of:
• a specificity segment, positioned 5′ of the PAM sequence, that hybridizes with nucleotides of a first target site of a target nucleic acid, and
• an orientation segment, positioned 3′ of the PAM sequence, that hybridizes with nucleotides of a second target site of the target nucleic acid;
• and
• (ii) a Cas9 guide RNA comprising a nucleotide sequence that hybridizes with the first target site of the target nucleic acid.