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US 12,371,709 B2Granted
Multifunctional nucleic acid reporter constructs
AssigneeDana-Farber Cancer Institute, Inc.
InventorsChristian Bowman-Colin, David M. Livingston, Laura C. Wong, Shweta Kitchloo
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Abstract
Nucleic acids comprising multifunctional double-stranded break reporter constructs for stable or transient transfection into a cell, as well as detecting a type of double-stranded break repair mechanism in a cell. Methods for detecting types of double-stranded break repair mechanism in a cell, as well as vectors and cells comprising nucleic acids comprising multifunctional double-stranded break reporter constructs.
Claims20
A nucleic acid comprising from 5′ to 3′:
- (a) a first cassette comprising:
- a 5′ portion of an open reading frame of a first reporter gene (5′RP1);
- a first microhomology domain (MHD);
- a sequence comprising from 5′ to 3′:
- a first double-stranded endonuclease recognition sequence (DSE-RS) recognized by a first double-stranded endonuclease (DSE), one or more transcriptional terminators, and a second DSE-RS recognized by a second DSE;
- a second MHD that has sequence homology to the first MHD;
- and
- a 3′ portion of an open reading frame for an allele of the first reporter gene (3′RP1),
- wherein a complete open reading frame of the first reporter gene (RP1) comprises from 5′ to 3′:
- 5′RP1, the first MHD or the second MHD, and 3′RP1,
- wherein removal of the sequence between the cleavage site in the first DSE-RS and the cleavage site in the second DSE-RS from the first cassette results in a reading frame shift of 3′RP1 relative to its native reading frame;
- (b) a second cassette comprising:
- an internal ribosome entry site sequence (IRES);
- an open reading frame of a second reporter gene (RP2) that is operably linked to the IRES;
- (c) a third cassette comprising a portion of an open reading frame of a third reporter gene (RP3), wherein the portion of RP3 comprises from 5′ to 3′:
- a first portion that has sequence homology to the 5′RP1;
- and
- a second portion that has sequence homology to the 3′RP1,
- wherein homologous recombination between the first and third cassettes results in a complete open reading frame of the third reporter gene comprising from 5′ to 3′:
- a 5′ portion of the 5′RP1, the portion of RP3, and a 3′ portion of the 3′RP1;
- and
- (d) a fourth cassette comprising an open reading frame of a fourth reporter gene (RP4) lacking a start codon,
- wherein the first and second cassettes are operably linked, and wherein the third and fourth cassettes are fused in the same reading frame.
20 more elements
A method for detecting a type of double-stranded break repair mechanism in an isolated cell, comprising:
- (i) integrating a nucleic acid sequence into the genome of a cell, wherein the nucleic acid sequence comprises at least four reporter genes that generate a different signal, wherein the nucleic acid comprises from 5′ to 3′:
- (a) a first cassette comprising:
- a 5′ portion of an open reading frame of a first reporter gene (5′RP1);
- a first microhomology domain (MHD);
- a sequence comprising from 5′ to 3′:
- a first double-stranded endonuclease recognition sequence (DSE-RS) recognized by a first double-stranded endonuclease (DSE), one or more transcriptional terminators, and a second DSE-RS recognized by a second DSE;
- a second MHD that has sequence homology to the first MHD;
- and
- a 3′ portion of an open reading frame for an allele of the first reporter gene (3′RP1),
- wherein a complete open reading frame of the first reporter gene (RP1) comprises from 5′ to 3′:
- 5′RP1, the first MHD or the second MHD, and 3′RP1,
- wherein removal of the sequence between the cleavage site in the first DSE-RS and the cleavage site in the second DSE-RS from the first cassette results in a reading frame shift of 3′RP1 relative to its native reading frame;
- (b) a second cassette comprising:
- an internal ribosome entry site sequence (IRES);
- an open reading frame of a second reporter gene (RP2) that is operably linked to the IRES;
- (c) a third cassette comprising a portion of an open reading frame of a third reporter gene (RP3), wherein the portion of RP3 comprises from 5′ to 3′:
- a first portion that has sequence homology to the 5′RP1;
- and
- a second portion that has sequence homology to the 3′RP1,
- wherein homologous recombination between the first and third cassettes results in a complete open reading frame of the third reporter gene comprising from 5′ to 3′:
- a 5′ portion of the 5′RP1, the portion of RP3, and a 3′ portion of the 3′RP1;
- and
- (d) a fourth cassette comprising an open reading frame of a fourth reporter gene (RP4) lacking a start codon,
- wherein the first and second cassettes are operably linked, and wherein the third and fourth cassettes are fused in the same reading frame;
- (ii) inducing a double-stranded break into the nucleic acid sequence;
- and
- (iii) detecting at least one signal generated by at least one reporter gene, that signal being indicative of the type of double-stranded break repair mechanism,
- wherein a different signal or combination of signals is detected for each of the double-stranded break repair mechanisms HR, NHEJ, MMEJ, and SSA.
25 more elements
Dependencies are inferred from the claim text.