Extrachromosomal dna labeling

The cell of any one of the preceding claims, wherein the gRNA comprises (a) a targeting sequence that is complementary to the breakpoint junction, (b) a RNA-guided nuclease-binding sequence, and (c) a Pumilio-FBF (PUF) domain binding sequence (PBS).

The cell of any one of the preceding claims, wherein the catalytically-inactive RNA-guided nuclease is dCas9.

The cell of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

A method comprising detecting the detectable molecule of the cell of any one of the preceding claims, thereby detecting the ecDNA.

A method comprising imaging the detectable molecule of the cell of any one of the preceding claims, thereby imaging the ecDNA.

A composition comprising the cell of any one of the preceding claims.

A method of producing the cell of any one of the preceding claims, the method comprising introducing into the cell (a) the catalytically-inactive RNA-guided nuclease or a nucleic acid encoding the catalytically-inactive RNA-guided nuclease, (b) a gRNA comprising (i) a targeting sequence that is complementary to the breakpoint junction, (ii) a RNA-guided nuclease-binding sequence, and (iii) Pumilio-FBF (PUF) domain binding sequence (PBS), and (c) a PUF domain linked to the detectable molecule, wherein the PUF domain binds to the PBS.

A molecular cytogenetic tracing method, comprising imaging ecDNA in a cell in which the ecDNA has been bound at a breakpoint junction by a catalytically-inactive RNA-guided nuclease complexed with a guide RNA (gRNA) linked to a detectable molecule.

The method of any one of the preceding claims, wherein the gRNA comprises (a) a targeting sequence that is complementary to the breakpoint junction, (b) a RNA-guided nuclease-binding sequence, and (c) a Pumilio-FBF (PUF) domain binding sequence (PBS).

The method of any one of the preceding claims, wherein the catalytically-inactive RNA-guided nuclease is dCas9.

The method of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

A molecular cytogenetic labeling method, the method comprising introducing into a cell comprising ecDNA (a) the catalytically-inactive RNA-guided nuclease, (b) a gRNA comprising (i) a targeting sequence that is complementary to the breakpoint junction, (ii) a RNA-guided nuclease-binding sequence, and (ii) a Pumilio-FBF (PUF) domain binding sequence (PBS), and (c) a PUF domain linked to the detectable molecule, wherein the PUF domain binds to the PBS.

The method of any one of the preceding claims, wherein the catalytically-inactive RNA-guided nuclease is dCas9.

The method of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

The method of any one of the preceding claims further comprising detecting the detectable molecule, thereby detecting the ecDNA.

The method of any one of the preceding claims further comprising imaging the detectable molecule, thereby imaging the ecDNA.

A cell comprising extrachromosomal deoxyribonucleic acid (ecDNA) that has been genetically modified at a breakpoint junction to express a detectable molecule.

The cell of any one of the preceding claims, wherein the breakpoint junction is genetically modified using a programmable nuclease-based gene editing system.

The cell of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

A method comprising detecting the detectable molecule of the cell of any one of the preceding claims, thereby detecting the ecDNA.

A method comprising imaging the detectable molecule of the cell of any one of the preceding claims, thereby imaging the ecDNA.

A composition comprising the cell of any one of the preceding claims.

A method of producing the cell of any one of the preceding claims, the method comprising introducing into the cell the programmable nuclease-based gene editing system of any one of the preceding claims.

A molecular cytogenetic tracing method, comprising imaging ecDNA in a cell in which the ecDNA that has been genetically modified at a breakpoint junction to express a detectable molecule.

The method of any one of the preceding claims, wherein the breakpoint junction is genetically modified using a programmable nuclease-based gene editing system.

The method of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

A molecular cytogenetic labeling method, the method comprising introducing into a cell comprising ecDNA a programmable nuclease-based gene editing system that targets a breakpoint junction in the ecDNA to incorporate a detectable molecule.

1. The cell of claim 5, wherein the cell comprises multiple PUF domains linked to multiple detectable molecules, and the PUF domains bind to the PBS.

The method of any one of the preceding claims, wherein the breakpoint junction is genetically modified using a programmable nuclease-based gene editing system.

The method of any one of the preceding claims, wherein the detectable molecule is a fluorescent protein.

The method of any one of the preceding claims further comprising detecting the detectable molecule, thereby detecting the ecDNA.

The method of any one of the preceding claims further comprising imaging the detectable molecule, thereby imaging the ecDNA.

A guide RNA (gRNA) comprising a targeting sequence that is complementary to a breakpoint junction in extrachromosomal DNA.

A composition or kit comprising the gRNA of any one of the preceding claims.

1. The method of claim 15, wherein the cell further comprises multiple PUF domains linked to multiple detectable molecules, and the PUF domains bind to the PBS.