Methods of screening using barcoded libraries

Patent metadata

Field	Value
Publication number	US-12157885-B2
Application number	US-201716315230-A
Country	US
Kind code	B2
Filing date	Jul 6, 2017
Priority date	Jul 6, 2016
Publication date	Dec 3, 2024
Grant date	Dec 3, 2024

Abstract

A method of screening is provided including providing a combination of a plurality of proliferating cell types wherein each proliferating cell type has a unique associated barcode within its genome that is different from other proliferating cell types of the plurality and wherein each proliferating cell type includes an exogenous gene that when expressed produces an associated phenotype to the proliferating cell type which alters proliferation of the cell type, introducing a perturbation to one or more of the plurality of proliferating cell types, inducing expression of one or more of the exogenous genes, determining the relative number of unique associated barcodes after a period of proliferation, and comparing the relative number of unique associated barcodes to a control relative number of unique associated barcodes to indicate the effect of the perturbation on the one or more of the plurality of proliferating cell types.

First claim

The invention claimed is:

1. A method of screening comprising
providing a combination of a plurality of proliferating cell types wherein each proliferating cell type has a unique associated barcode within its genome that is different from other proliferating cell types of the plurality and wherein each proliferating cell type includes an exogenous gene present on a plasmid that when expressed produces an associated phenotype to the proliferating cell type which alters proliferation of the cell type,
introducing a perturbation to one or more of the plurality of proliferating cell types,
inducing expression of one or more of the exogenous genes,
determining the relative number of unique associated barcodes after a period of proliferation, and
comparing the relative number of unique associated barcodes to a control relative number of unique associated barcodes to indicate the effect of the perturbation on the one or more of the plurality of proliferating cell types.

2. The method of claim 1 wherein expression of the exogenous gene produces an associated phenotype to the proliferating cell type which reduces proliferation of the cell type.

3. The method of claim 1 wherein the exogenous gene is a toxicity gene that when expressed causes toxicity to the proliferating cell type thereby reducing proliferation of the proliferating cell type.

4. The method of claim 3 wherein the exogenous gene encodes an aggregation prone protein hnRNPA1 or FUS.

5. The method of claim 1 wherein the perturbation is a chemical perturbation.

6. The method of claim 1 wherein the perturbation is a genetic perturbation.

7. The method of claim 1 wherein the control relative number of unique associated barcodes is determined by providing a combination of a plurality of proliferating cell types wherein each proliferating cell type has a unique associated barcode that is different from other proliferating cell types of the plurality and wherein each proliferating cell type has an exogenous gene that when expressed produces an associated phenotype to the proliferating cell type which alters proliferation of the proliferating cell type,
inducing expression of one or more of the exogenous genes, and
determining the control relative number of unique associated barcodes after a period of proliferation.

8. The method of claim 1 wherein the perturbation is accomplished by introduction of a candidate compound to the combination of the plurality of proliferating cell types.

9. The method of claim 1 wherein the perturbation is accomplished by introduction of a candidate drug compound to the combination of the plurality of proliferating cell types, wherein the candidate drug compound inhibits the associated phenotype.

10. The method of claim 1 wherein the perturbation is accomplished by inducing expression of a target gene within the combination of the plurality of proliferating cell types.

11. The method of claim 1 wherein the perturbation is accomplished by reducing expression of a target gene within the combination of the plurality of proliferating cell types.

12. The method of claim 1 wherein the relative number of unique associated barcodes is determined by sequencing of the genome of the plurality of proliferating cell types.

13. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of yeast strains.

14. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of bacterial strains.

15. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of mammalian cell lines.

16. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of human cell lines.

17. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of neuronal cell lines.

18. The method of claim 1 wherein the plurality of proliferating cell types is a plurality of human neuronal cell lines.

19. The method of claim 1 wherein the proliferating cell type is a proliferating organism or cell line.

20. The method of claim 1 wherein the proliferating cell type is a member selected from the group consisting of HEK 293, Chinese Hamster Ovary cells, HEK 293F, HEK 293H, HEK 293A, HEK 293 FT, HEK293T, CHO DG44, CHO-S, CHO-DXB11, Expi293F, ExpiCHO-S, T-Rex, Hela, MCF7, COS7, NIH 3T3, U2OS, A375, A549, N2A, PGP1 iPS, BHK, Hap1, Jurkat, and N0.

21. The method of claim 1 wherein the exogenous gene is a member selected from the group consisting of Abeta, Androgen receptor (AR), a-syn A30P, a-syn A53T, a-syn WT, ataxin1, Ataxin1 [Q84], ataxin3, ATXN7, C9orf72 GA100, C9orf72 GA200, C9orf72 GA50, C9orf72 GR100, C9orf72 GR50, C9orf72 PR50, CHOPS M8, CHOPS Wt, EWSR1, EWSR1c1655t, EWSR1g1532c, EWSR1g1750a, FUS WT, FUS-P525L, hnRNPA1 WT, hnRNPA1D262V, hnRNPA2B1 D290V, hnRNPA2B1WT, htt72Q, htt103Q, Htt46Q, PABPN1, SOD1 A4V, SOD1 G85R, SOD1 G93A, SOD1 WT, TAF15, TAF15c1222t, TAF15g1172a, Tau, TDP43, TDP-43 G294A, TDP-43 M337V, TDP-43 Q331K, UBQLN2, CHMP2B, PABPN1, ARX, SOX3, RUNX2, ZIC2, PHOX2B, HOXD13, HOXA13, FOXL2, ATXN2, CACNA1A, PrP, and TBP.

22. The method of claim 1 wherein introducing the perturbation comprises administering Lovastatin or Celestrol.

23. The method of claim 1 wherein introducing the perturbation comprises providing a Cas protein and a guide RNA targeting an endogenous gene.

Assignees

• Harvard College
• Massachusetts Gen Hospital

Inventors

• Kaas Christian Schroeder
• Chavez Alejandro

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/905

  in yeast · CPC title