IP Library › Granted Patent US 12,516,373
Granted Patent B2
US 12,516,373 · App. 17/817,516 · Granted Jan 6, 2026

In situ readout of DNA barcodes

Inventors: Amjad Askary (Pasadena, CA); Michael B. Elowitz (Los Angeles, CA); Mark W. Budde (Arcadia, CA); Carlos Lois (South Pasadena, CA); Luis Sanchez Guardado (Pasadena, CA); Long Cai (Pasadena, CA); James Linton (Pasadena, CA)
Assignee: California Institute of Technology
C12Q1/6874C12Q1/6818C12Q1/6876G01N1/30
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Quick Facts
Patent No.
US 12,516,373
App. No.
17/817,516
Granted
Jan 6, 2026
Kind
B2
Abstract

Disclosed herein include systems, methods, compositions, and kits for in situ readout of barcodes, such as DNA barcodes. Barcode constructs containing a promoter (e.g., a phage promoter) that is inactive in live cells can be integrated in the genomes of cells. Cells can be fixed, and phage RNA polymerase can be used for transcription of the barcode to RNA transcripts. The RNA transcripts can be detected using, for example, fluorescent imaging and used to determine barcode sequences.

Claims (29)

1 . A method of determining barcode sequences in situ, comprising:

providing a plurality of cells each comprising a barcode polynucleotide with a barcode sequence;

fixing the plurality of cells using a fixative to generate a plurality of fixed cells;

generating a plurality of barcode molecules comprising the barcode sequence of the barcode polynucleotide in each of the plurality of fixed cells;

contacting the plurality of fixed cells with a plurality of detection probes each comprising a barcode binding sequence and an initiator sequence, thereby each of the plurality of barcode molecules comprising the barcode sequence of the barcode polynucleotide in the fixed cell hybridizes to a detection probe, of the plurality of detection probes, comprising the barcode binding sequence reverse complementary to the barcode sequence, or a portion thereof;

contacting the plurality of fixed cells with a plurality of first amplifier probes each comprising a different fluorophore, thereby a first amplifier probe of the plurality of first amplifier probes hybridizes to (i) the initiator sequence of a detection probe of the plurality of detection probes hybridized to a barcode molecule in a fixed cell of the plurality of fixed cells;

detecting the fluorophore, or fluorescence thereof, of the first amplifier probe hybridized to the detection probe hybridized to the barcode molecules in each of the plurality of fixed cells using fluorescence imaging; and

determining the barcode sequence in each of the plurality of fixed cells using the fluorophore detected, wherein the fluorophore detected indicates the barcode sequence of the barcode polynucleotide in the one or more fixed cells.

2 . The method of claim 1 , wherein providing the plurality of cells comprises: integrating the barcode polynucleotide into a genome of one, at least one, or each of the plurality of cells.

3 . The method of claim 2 , wherein integrating the barcode polynucleotide comprises: integrating the barcode polynucleotide into the genome of one, at least one, or each of the plurality of cells at a specific site of the genome.

4 . The method of claim 2 , wherein integrating the barcode polynucleotide into the genome of one, at least one, or each of the plurality of cells comprises: transfecting the cell with a donor plasmid comprising the barcode polynucleotide, a sequence thereof, a subsequence thereof, of a reverse complementary sequence of any of the preceding.

5 . The method of claim 2 , wherein integrating the barcode polynucleotide comprises: integrating the barcode polynucleotide into the genome of one, at least one, or each of the plurality of cells using a viral vector, wherein the viral vector comprises a polynucleotide comprising the barcode polynucleotide, a sequence thereof, a subsequence thereof, or a reverse complementary sequence of any of the proceeding.

6 . The method of claim 1 , wherein the barcode polynucleotide of one, at least one, or each of the plurality of cells comprises barcode sequences, and wherein a combination of the barcode sequences is selected from at least 500000, possible combinations of the barcode sequences.

7 . The method of claim 6 , wherein the barcode sequences are downstream of at least one promoter, and/or wherein two of the barcode sequences are downstream of different promoters, optionally wherein the different promoters comprise an identical promoter sequence.

8 . The method of claim 1 , wherein the barcode polynucleotides of at least two cells of the plurality of cells comprise an identical barcode sequence.

9 . The method of claim 1 , wherein the barcode polynucleotides of at least two cells of the plurality of cells comprise different barcode sequences.

10 . The method of claim 8 , wherein the at least two cells are cells of a cell type, cells of a cell subtype, and/or cells of an identical lineage.

11 . The method of claim 8 , wherein the at least two cells are cells of different cell types, cells of different cell subtypes, and/or cells of different lineages.

12 . The method of claim 8 , wherein a first cell of the at least two cells is a cell of interest, and/or wherein a second cell of the at least two cells is not a cell of interest.

13 . The method of claim 1 , wherein generating the plurality of barcode molecules comprises: transcribing the barcode polynucleotide in each of the plurality of fixed cell to generate the plurality of barcode molecules comprising the barcode sequence of the barcode polynucleotide in the fixed cell.

14 . The method of claim 1 , comprising: determining lineages of, and/or a clonal relationship between, two or more fixed cells of the plurality of fixed cells using the barcode sequence of the barcode polynucleotide in each of the two or more fixed cells.

15 . The method of claim 1 , comprising:

determining a spatial relationship of two or more fixed cells of the plurality of fixed cells; and

correlating the barcode sequences of the barcode polynucleotide in each of the two or more fixed cells with a spatial relationship of the two or more fixed cells.

16 . The method of claim 1 , comprising: correlating gene expressions of two or more fixed cells of the plurality of fixed cells with the lineages of, the clonal relationship between, and/or the spatial relationship of, the two or more fixed cells.

17 . The method of claim 1 , wherein the plurality of cells is from a sample comprising a cell culture, a tissue, an organ, an embryo, an organism, a section thereof.

18 . The method of claim 1 , wherein the plurality of cells is from a sample comprising an in vivo sample and/or an in vitro sample.

19 . The method of claim 1 , wherein the plurality of cells comprises one or more tumor cells, one or more immune cells, one or more epithelial cells, one or more nervous cells, one or more blood cells, one or more bone cells, one or more fat cells, one or more muscle cells, and/or one or more sex cells.

20 . The method of claim 1 , wherein two, at least two, or each of the plurality of cells are cultured under an identical condition, and wherein the identical condition or each of the different conditions comprises a genetic perturbation, an environmental perturbation, or a combination thereof.

Assignments (4)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 5, 2022
From: ASKARY, AMJAD
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 060733/0680 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 5, 2022
From: ELOWITZ, MICHAEL B.
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 060733/0707 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 5, 2022
From: BUDDE, MARK W.; LOIS, CARLOS; GUARDADO, LUIS SANCHEZ; CAI, LONG; LINTON, JAMES
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 060733/0716 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 5, 2022
From: HOWARD HUGHES MEDICAL INSTITUTE
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 060733/0736 →
Continuity (4)
Continuation 16701087 · Dec 2, 2019
Provisional Application 62936307 · Nov 15, 2019
Provisional Application 62774754 · Dec 3, 2018
Related Publication 20220396833A1 · Dec 15, 2022
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