Granted Patent
B2
US 12,612,657 · App. 16/992,815 · Granted Apr 28, 2026
Extrachromosomal DNA labeling
Assignee:
The Jackson Laboratory
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Abstract
Provided herein are methods and tools for targeting detecting (e.g., imaging) extrachromosomal DNA, for example, in cancer cells.
Claims (15)
1 . A method capable of detecting a single extrachromosomal deoxyribonucleic acid (ecDNA) in a cell, the method comprising binding at most one non-repetitive breakpoint junction sequence on the ecDNA with a catalytically-inactive RNA-guided nuclease complexed with a guide RNA (gRNA) targeting the non-repetitive breakpoint junction sequence and linked to a multiplicity of detectable molecules that are sufficient to be detected.
2 . The method of claim 1 , wherein the cell is a cancer cell.
3 . The method of claim 2 , wherein the cancer cell is a glioblastoma cell, a melanoma cell, a sarcoma cell, a bladder cancer cell, or an esophageal cancer cell.
4 . The method of claim 1 , wherein the gRNA comprises (a) a targeting sequence that is complementary to the non-repetitive breakpoint junction, (b) a RNA-guided nuclease-binding sequence, and (c) multiple Pumilio-FBF domain binding sequences (PBS).
5 . The method of claim 4 , wherein the multiple detectable molecules are linked to the gRNA via multiple Pumilio-FBF (PUF) domains, and wherein the multiple PUF domains bind to the PBS on the gRNA.
6 . The method of claim 1 , wherein the catalytically-inactive RNA-guided nuclease is dCas9.
7 . The method of claim 1 , wherein the detectable molecules are fluorescent proteins.
8 . The method of claim 1 , wherein the detecting comprises imaging the detectable molecules of the cell.
9 . The method of claim 1 , further comprising introducing into the cell:
(a) the catalytically-inactive RNA-guided nuclease or a nucleic acid encoding the catalytically-inactive RNA-guided nuclease,
(b) the gRNA comprising:
(i) a targeting sequence that is complementary to the non-repetitive breakpoint junction,
(ii) a RNA-guided nuclease-binding sequence, and
(ii) Pumilio-FBF domain binding sequences (PBS), and
(c) one or more Pumilio-FBF (PUF) domains linked to the multiplicity of detectable molecules, wherein the one or more PUF domains bind to the PBS.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 13, 2021
From: YI, EUNHEE; VERHAAK, ROEL
To: THE JACKSON LABORATORY
Reel/Frame 057456/0862 →
Continuity (2)
Provisional Application
62887931
· Aug 16, 2019
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