LABELING OF DNA
Methods of labeling DNA molecules comprising the use of one or more dCas proteins and one or more labeled guide RNAs (gRNAs) are described herein. In some embodiments, the labeled DNA molecules are elongated in fluidic nanochannels, where they can be analyzed.
1 . A method of labeling a DNA at a target sequence, comprising:
contacting a DNA comprising a first target sequence with:
a first dCas protein; and
a first labeled guide RNA (gRNA) comprising a first crRNA and a first tracrRNA comprising a first label, wherein the first crRNA is complementary to the first target sequence or a portion thereof;
incubating the first DNA, the first dCas protein, and the first labeled gRNA, whereby the first dCas protein, the DNA, and the first labeled gRNA form a complex wherein the first crRNA is hybridized to the first target sequence or the portion thereof,
thereby labeling the DNA at the first target sequence to form a labeled DNA.
2 . The method of claim 1 , wherein the DNA comprises a second target sequence and the method further comprises:
contacting the DNA with:
a second dCas protein; and
a second labeled gRNA comprising a second crRNA and a second tracrRNA comprising a second label that is different from the first label, wherein the second crRNA is complementary to a second target sequence or a portion thereof; and
incubating the DNA with the second dCas protein, the second labeled gRNA, wherein the second dCas protein, the DNA, and the second labeled gRNA form a second complex, wherein the second crRNA is hybridized to the second target sequence or the portion thereof,
thereby labeling the DNA at the second target sequence with the second label.
3 . The method of claim 2 , wherein the DNA is contacted with the first labeled gRNA and the second labeled gRNA at the same time, and wherein the DNA is incubated with the first dCas protein, the second dCas protein, the first labeled gRNA, and the second labeled gRNA at the same time.
4 . The method of claim 1 , wherein the DNA remains intact throughout the method.
5 . The method of claim 1 , wherein at least one of the first and second target sequences (a) is in a region comprising multiple repeats or structural variants not amenable to enzymatic motif labeling, (b) is in a region predicted to form or susceptible of forming a fragile site upon nick translation labeling, or (c) is comprised by a genomic region that does not comprise unevenly distributed labels upon nick labeling.
6 . (canceled)
7 . (canceled)
8 . The method of claim 1 , further comprising selecting the first target sequence, the second target sequence, or both as in a region comprising multiple repeats or structural variants not amenable to enzymatic motif labeling
9 . The method of claim 5 , wherein the enzymatic motif labeling is nickase labeling.
10 . The method of claim 1 , wherein at least one of the first and second target sequences is selected as in a region predicted to form a fragile site upon nick translation labeling.
11 . (canceled)
12 . (canceled)
13 . The method of claim 1 , further comprising linearizing the labeled DNA in a fluidic nanochannel, wherein the DNA remains intact upon said linearizing.
14 . The method of claim 13 , further comprising detecting a relative distance between the first label and the second label on the linearized DNA in the fluidic nanochannel.
15 . (canceled)
16 . (canceled)
17 . (canceled)
18 . The method of claim 1 , wherein the first dCas protein, the second dCas protein, or both comprise one or more mutations and/or one or more deletions in a HNH domain and/or RuvC-like domain.
19 . (canceled)
20 . The method of claim 1 , wherein the first dCas protein is not labeled
21 . The method of claim 1 , wherein the second dCas protein is not labeled
22 . The method of claim 1 , wherein the first and/or the second crRNA comprises a sequence of about 10-40 nucleotides that is complementary to the first and/or the second target sequence.
23 . (canceled)
24 . The method of claim 1 , further comprising labeling the DNA by direct enzyme labeling, or nick labeling.
25 . (canceled)
26 . (canceled)
27 . The method of claim 24 , wherein the nick labeling comprises nicking the DNA followed by labeling of the nicks without repairing the labeled DNA.
28 . The method of claim 24 , wherein the nick labeling comprises nicking the DNA followed by labeling of the nicks on the DNA and repairing the labeled DNA.
29 . (canceled)
30 . The method of claim 1 , wherein the first label and/or the second label is selected from the group consisting of: a fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a hapten, a streptavidin, an avidin, a neutravidin, a biotin, and a reactive group a peptide, a protein, a magnetic bead, a radiolabel, a non-optical label, and a combination thereof.
31 . (canceled)
32 . A DNA composition comprising:
a DNA molecule;
a first dCas protein; and
a first labeled guide RNA (gRNA) comprising a first crRNA and a first tracrRNA comprising a label,
wherein the first dCas protein, the first labeled gRNA, and the first DNA molecule are comprised by a complex comprising the first crRNA hybridized to a first target sequence of the DNA molecule or a portion thereof.
33 . The DNA composition of claim 32 , further comprising:
a second dCas protein
a second labeled gRNA comprising a second cRNA and a second tracrRNA labeled with a second label different from the first label,
wherein the second dCas protein, the second labeled gRNA, and the DNA are comprised by a complex comprising the second crRNA hybridized to a second target sequence of the DNA molecule or a portion thereof.
34 .- 55 . (canceled)