Granted Patent B2

US 12,529,092 · App. 17/426,453 · Granted Jan 20, 2026

Inventors: Aviv Regev (Cambridge, MA); Sanja Vickovic (Cambridge, MA)

Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology

C12Q1/6841C12N9/22C12N15/1096C12N15/11C12N2310/20C12N2800/80

Abstract

Claims (53)

1 . A method of spatiotemporal processing of a sample of a plurality of cells comprising:

a. depositing a plurality of spatial barcodes on a solid substrate, the spatial barcodes each defining an x,y position on the solid substrate and further comprising a capture molecule;

b. depositing the sample of the plurality of cells on the surface of the solid substrate;

c. capturing material from one or more cells of the plurality of cells with the capture molecule of the spatial barcode, thereby linking the captured material from the one or more cells with the spatial barcode, optionally wherein the captured material comprises nucleic acids, proteins or any combination thereof; and

d. correlating the captured material to the x,y positions in the sample on the solid substrate, wherein the x,y positions have a resolution of 5 μm or less.

2 . The method of claim 1 , further comprising capturing an image of the sample on the solid substrate, and optionally annotating regions of the image of the sample optionally based on morphology.

3 . The method of claim 2 , wherein correlating further comprises assigning pixel coordinates to the image and coordinating to the x,y position of the spatial barcode.

4 . The method of claim 3 , further comprising assigning a cell type to cells in the sample.

5 . The method of claim 1 , further comprising

d. staining the sample;

e. recording the morphology of the stained sample;

f. permeabilizing the sample;

g. capturing mRNA of the sample with the capture molecule, thereby linking mRNA of the cells of the sample with the spatial barcode;

h. preparing a library of cDNA molecules from the captured mRNA and the linked spatial barcode; and optionally

i. ablating a single layer of the plurality of cells and performing the step of capturing material from one or more cells of the plurality of cells in a second layer of the cells.

6 . The method of claim 5 , wherein one or more of steps d-i are performed on a fully automated spatial transcriptomics platform.

7 . The method of claim 5 , wherein the step of recording the morphology comprises automated capturing of a plurality of images and image stitching.

8 . The method of claim 5 , further comprising sequencing the library of cDNA molecules and correlating each of the cDNA molecules to a position in the sample on the solid substrate.

9 . The method of claim 8 , further comprising assigning a cell type or cell subtype to the plurality of cells in the sample, the assigning comprising detecting differential expression of the cDNA molecules to generate a gene signature and identifying cell type based on the gene signature at positions in the sample.

10 . The method of claim 8 , further comprising integrating the position of the cDNA molecules with images generated by automated capturing of a plurality of images and image stitching.

11 . The method of claim 1 , further comprising staining the plurality of cells, wherein staining optionally comprises fluorescent or bright field staining.

12 . The method of claim 1 , further comprising depositing a plurality of CRISPR-Cas systems on the solid substrate, wherein each of the CRISPR-Cas systems comprises (i) a CRISPR-Cas protein or one or more nucleic acid sequences encoding the CRISPR-Cas protein, and (ii) a guide sequence capable of hybridizing with a target sequence.

13 . The method of claim 12 , wherein one or more CRISPR-Cas systems are deposited at each defined x,y position on the solid substrate.

14 . The method of claim 12 , wherein the guide sequence is linked to the spatial barcode.

15 . The method of claim 1 , further comprising delivering CRISPR-Cas systems to the sample prior to or subsequent to depositing the sample on the solid substrate.

16 . The method of claim 1 , wherein the spatial barcode is provided in a droplet.

17 . The method of claim 16 , wherein the droplet comprises a plurality of spatial barcodes.

18 . The method of claim 1 , wherein the spatial barcode comprises a bead.

19 . The method of claim 18 , wherein the bead comprises a plurality of spatial barcodes.

20 . The method of claim 18 , wherein the bead comprises color-coded beads.

21 . The method of claim 18 , wherein the bead comprises a conductivity-coded bead, optionally wherein the conductivity-coded bead is deposited on the solid substrate, the solid substrate comprising pre-etched wells.

22 . The method of claim 1 , further comprising the step of decoding the spatial barcode, the decoding comprising sequential hybridization, in-situ sequencing, laser scanning, DNA microscopy, FISH, smFISH, or in situ PCR.

23 . The method of claim 1 , further comprising sequencing the captured material.

24 . The method of claim 1 , further comprising releasing the captured material, optionally wherein the spatial barcode comprises a cleavable linker.

25 . The method of claim 24 , wherein the releasing comprises the release of spatially positioned barcodes into the tissue with the addition of polymer, betaine and/or MgCl 2 .

26 . The method of claim 24 , wherein the cleavable linker is a restriction site, and releasing the captured material comprises utilizing a restriction enzyme specific to the restriction site, and cleaving the captured molecule.

27 . The method of claim 1 , wherein the spatial barcode is an oligonucleotide and the captured material comprises nucleic acids, and the method further comprises synthesizing a complementary strand to the spatial barcode or captured material using a polymerase, and releasing the complementary strand or the spatial barcode and captured material.

28 . The method of claim 1 , wherein the plurality of cells is a tissue sample, optionally a mammalian tissue sample.

29 . A method of spatiotemporal processing of a sample of a plurality of cells comprising:

a. depositing a plurality of spatial barcodes on a solid substrate, the spatial barcodes each defining an x,y position on the solid substrate and further comprising a capture molecule;

b. depositing the sample of the plurality of cells on the surface of the solid substrate, wherein the plurality of cells is a tissue sample, optionally a mammalian tissue sample, and wherein the tissue sample is greater than about 0.5 cm in thickness; and

c. capturing material from one or more cells of the plurality of cells with the capture molecule of the spatial barcode, thereby linking the captured material from the one or more cells with the spatial barcode, optionally wherein the captured material comprises nucleic acids, proteins or any combination thereof.

30 . The method of claim 28 , wherein the tissue sample is a biopsy sample.

31 . The method of claim 28 , wherein the tissue sample is from the central nervous system.

32 . The method of claim 1 , wherein the solid substrate comprises a glass slide, a polymer, or an imaging fiber.

33 . The method of claim 1 , wherein the solid substrate comprises an array of microwells, the microwells each about 2 um, optionally with a 3 um distance from center to center of each well.

34 . The method of claim 1 , wherein the depositing comprises inkjet, contact printing or Fluorescent Activated Cell Sorting (FACS).

35 . The method of claim 1 , wherein the depositing is random or ordered.

36 . The method of claim 1 , wherein depositing the spatial barcode comprises the binding of the spatial barcode to the solid substrate, optionally wherein the binding of the spatial barcode to the solid substrate is covalent or non-covalent bonding.

37 . The method of claim 36 , wherein the solid substrate comprises a surface with available active groups that facilitate the bonding of the spatial barcode to the solid substrate surface.

38 . The method of claim 1 , wherein the spatial barcode comprises an oligonucleotide sequence, optionally wherein the oligonucleotide sequence further comprises one or more of a unique molecular identifier (UMI), an adapter sequence, and a primer sequence.

39 . The method of claim 38 , further comprising building the spatial barcode on the solid substrate, optionally wherein the building the spatial barcode comprises bridge PCR or solid extension or comprises distributing oligonucleotide sequences on the solid substrate, adding padlock probes, and amplifying and decoding the oligonucleotides on the surface.

40 . The method of claim 1 , wherein the capture molecule comprises a Tn5 sequence, a 16S sequence, a poly (d) T sequence, a random hexamer sequence, a trypsin molecule, an antibody, a Protein Epitope Signature Tag (PrEST) sequence, or a combination thereof.

Assignments (4)

Continuity (3)

References Cited (190)