Granted Patent B2

US 12,281,301 · App. 17/048,369 · Granted Apr 22, 2025

Inventors: Feng Zhang (Cambridge, MA); Jonathan Leo Schmid-Burgk (Cambridge, MA); Veit Hornung (Cambridge, MA)

Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY

C12N15/1065C12N9/22C12N15/65C12N2310/20

Abstract

Claims (42)

1. A method of determining the distribution of protein levels in a population of cells, said method comprising:

a) sorting a library of cells into at least two groups based on expression of the detectable marker in each cell; and

b) nucleic acid sequencing of the cells in each group, wherein the tagged target genes in each group are determined;

wherein the library of cells comprises a plurality of cells, wherein each cell comprises a polynucleotide sequence encoding a protein tag integrated into the genome of the cell in frame with a protein coding gene selected from a set of target genes, wherein the library comprises more than one cell tagged at each target gene;

wherein the library of cells is treated with a perturbation prior to determining expression of proteins, wherein the perturbation comprises a small molecule, protein, RNAi, CRISPR system, TALE system, Zn finger system, meganuclease, pathogen, allergen, recombinant virus, temperature, salt, lipid, biomolecule, a pool of any perturbation thereof, or any combination thereof; and

wherein the protein tag is a detectable marker and the cell library is for use in detecting the distribution of protein levels in single cells comprising a plurality of cells.

2. The method according to claim 1 , wherein the sequencing comprises PCR amplification of the UMIs with primers specific to the polynucleotide sequence encoding a detectable marker; or

wherein the sequencing comprises transcription of the tagged gene by T7 polymerase, cDNA production, and sequencing of the cDNA; or

wherein the sequencing comprises tagmentation with Tn5, PCR amplification; and/or

wherein the localization of proteins is determined by further sorting the cells based on cleavage of the cleavable marker by the protease localized to a cellular compartment; or wherein the localization of proteins is determined by comparing the distribution of protein levels of tagged genes between sorted cells and sorted nuclei obtained from the library.

3. The method of claim 1 for use in identifying cell cycle regulated proteins; or

for use in identifying or confirming drug targets that are not regulated at the transcript level.

4. The method of claim 1 , further comprising transferring the library to an in vivo model and recovering the cells before the step of sorting.

5. The method of claim 1 , wherein the cell library is for use in detecting protein interactions between a protein of interest and a set of target proteins, wherein each cell comprises a first polynucleotide sequence encoding a first protein tag that is a complementary protein integrated into the genome of the cell in frame with the protein of interest or comprises a first polynucleotide sequence encoding a fusion protein of the first complementary protein and protein of interest, wherein each cell comprises a second polynucleotide sequence encoding a second protein tag that is a complementary protein integrated into the genome of the cell in frame with a protein coding gene selected from the set of target genes, wherein the library comprises more than one cell tagged at a target gene, whereby an interaction between the protein of interest and a target gene can be detected with a detectable marker.

6. The method of claim 5 , wherein the second polynucleotide sequence further comprises a codon-neutral unique molecular identifier (UMI) sequence or a non-coding UMI after the detectable marker coding sequence.

7. The method of claim 6 , wherein the library is sequence-verified, such that each UMI identifies a tagged target gene.

8. The method of claim 7 , wherein:

a) the first and second complementary proteins comprise protein complementary assay (PCA) fragments, preferably, wherein the PCA fragments comprise split fluorescent protein fragments or split TEV fragments; or

b) one of the first or second complementary proteins comprises a permuted inactive reporter and the other complementary protein comprises TEV; or

c) the first and second complementary proteins comprise a different epitope tag, whereby interaction may be detected by proximity ligation; or

d) one of the first or second complementary proteins comprises one or more TEV cleavage sites followed by one or more epitopes and the other complementary protein comprises TEV.

9. The method of claim 6 , wherein the second polynucleotide sequence comprises a selectable marker operably linked to a separate regulatory element; or wherein the polynucleotide sequence comprises an IRES or a 2A peptide, whereby the selectable marker is expressed as a separate protein; and/or

wherein the second polynucleotide sequence comprises a T7 RNA polymerase promoter;

and/or wherein each cell comprises a sequence encoding a guide sequence specific for the tagged target gene, whereby detection of the sequence indicates the tagged target gene; and/or

wherein the library comprises eukaryotic cells; and/or

wherein the cells of the library were generated from cells configured to express a CRISPR enzyme; or wherein the cells of the library were generated from cells obtained from a transgenic animal configured to express a CRISPR enzyme.

10. The method of claim 1 , wherein the cell library is for use in detecting protein modifications comprising a plurality of cells, wherein each cell stably expresses a protein tag that is a fusion protein comprising a protein modification binding protein fused to a first complementary protein, wherein each cell comprises a polynucleotide sequence encoding a second protein tag that is a complementary protein integrated into the genome of the cell in frame with a protein coding gene selected from the set of target genes, wherein the library comprises more than one cell tagged at each target gene and wherein binding of a protein modification binding protein to a target gene protein can be detected with a detectable marker.

11. The method of claim 10 , wherein the polynucleotide sequence further comprises a codon-neutral unique molecular identifier (UMI) sequence or a non-coding UMI after the detectable marker coding sequence.

12. The method of claim 10 , wherein:

a) the first and second complementary proteins comprise protein complementary assay (PCA) fragments, preferably, wherein the PCA fragments comprise split fluorescent protein fragments or split TEV fragments; or

b) one of the first or second complementary proteins comprises a permuted inactive reporter and the other complementary protein comprises TEV; or

c) the first and second complementary proteins comprise a different epitope tag, whereby protein modification may be detected by proximity ligation; or

d) one of the first or second complementary proteins comprises one or more TEV cleavage sites followed by one or more epitopes and the other complementary protein comprises TEV.

13. The method of claim 10 , wherein the polynucleotide sequence comprises a selectable marker operably linked to a separate regulatory element; and/or

wherein the polynucleotide sequence comprises a T7 RNA polymerase promoter; and/or

wherein each cell comprises a sequence encoding a guide sequence specific for the tagged target gene, whereby detection of the sequence indicates the tagged target gene; and/or

wherein the library comprises eukaryotic cells; and/or

wherein the cells of the library were generated from cells configured to express a CRISPR enzyme; or the cells of the library were generated from cells obtained from a transgenic animal configured to express a CRISPR enzyme.

14. The method according to claim 2 , wherein the PCR amplification comprises a nested PCR and sequencing the PCR products.

15. The method according to claim 2 , comprising linear amplification (LAM).

16. The method according to claim 2 , wherein the PCR amplification comprises nested PCR and sequencing of the amplified DNA.

17. The method according to claim 2 , wherein the nuclei are fixed.

Assignments (4)

Continuity (3)

References Cited (22)