Granted Patent B2

US 12,441,995 · App. 17/027,151 · Granted Oct 14, 2025

Inventors: Feng Zhang (Cambridge, MA); Ophir Shalem (Cambridge, MA); Neville Espi Sanjana (Cambridge, MA); John Doench (Cambridge, MA); David Root (Cambridge, MA); Ella Nicole Biewener Hartenian (Cambridge, MA)

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

C12N15/1093C12N9/22C12N15/102C12N15/1082C12N15/63C12N15/907C12N2310/20

Abstract

Claims (29)

1. A method for identifying one or more genomic loci associated with drug resistance, comprising:

contacting a population of cells with a composition comprising one or more vectors that encode (a) a Cas9 protein and (b) a plurality of CRISPR-Cas system guide RNAs targeting a plurality of different genomic loci, wherein the plurality of CRISPR-Cas system guide RNAs comprise guide RNAs encoded by plasmid library of ATCC Deposit No. PTA121339, guide RNAs encoded by plasmid library of ATCC Deposit No. 121340, guide RNAs encoded by plasmid library of ATCC Deposit No. 121341, guide RNAs encoded by plasmid library of ATCC Deposit No. 121342, guide RNAs encoded by plasmid library of ATCC Deposit No. 121343, or a combination thereof,

selecting cells successfully transduced with the vectors, wherein the selected cells each comprises a CRISPR-Cas system comprising the Cas9 protein and one of the guide RNAs;

treating the selected cells with a drug to obtain cells having resistance to the drug; and

profiling the depletion or enrichment of the guide RNAs in the cells having resistance to the drug by high-throughput sequencing to identify one or more genomic loci associated with the resistance to the drug.

2. The method of claim 1 , wherein the cells are eukaryotic cells.

3. The method of claim 2 , wherein the cells are mammalian cells or human cells.

4. The method of claim 3 , wherein the cells are cancer cells or stem cells.

5. The method of claim 1 , wherein the cells are transduced with a multiplicity of infection (MOI) of 0.3-0.75.

6. The method of claim 1 , wherein the cells are transduced with a multiplicity of infection (MOI) of 0.3-0.4.

7. The method of claim 1 , wherein the Cas9 is Streptococcus pyogenes Cas9.

8. The method of claim 1 , wherein the Cas9 is Staphylococcus aureus Cas9.

9. The method of claim 1 , wherein the Cas9 and the guide RNAs are encoded in the same vector.

10. The method of claim 1 , wherein the Cas9 and the guide RNAs are encoded in different vectors.

11. The method of claim 1 , wherein the vectors are lentivirus, adenovirus, or adeno-associated virus vectors.

12. The method of claim 1 , wherein the composition comprises vectors encoding 100 or more different guide RNAs.

13. The method of claim 1 , wherein the composition comprises vectors encoding 1,000 or more different guide RNAs.

14. The method of claim 1 , wherein the composition comprises vectors encoding 20,000 or more different guide RNAs.

15. The method of claim 1 , wherein the drug is a cancer drug.

16. A method for identifying one or more genomic loci associated with cell survival comprising:

contacting a population of cells with a composition comprising one or more vectors that encode (a) a Cas9 protein and (b) a plurality of CRISPR-Cas system guide RNAs targeting a plurality of different genomic loci, wherein the plurality of CRISPR-Cas system guide RNAs comprise guide RNAs encoded by plasmid library of ATCC Deposit No. PTA121339, guide RNAs encoded by plasmid library of ATCC Deposit No. 121340, guide RNAs encoded by plasmid library of ATCC Deposit No. 121341, guide RNAs encoded by plasmid library of ATCC Deposit No. 121342, guide RNAs encoded by plasmid library of ATCC Deposit No. 121343, or a combination thereof,

selecting cells successfully transduced with the vectors, wherein the selected cells each comprises a CRISPR-Cas system comprising the Cas9 protein and one of the guide RNAs;

applying an selective pressure to the selected cells to obtain cells survived under the selective pressure; and

profiling the depletion or enrichment of the guide RNAs in the cells survived under the selective pressure by high-throughput sequencing to identify one or more genomic loci associated with survival under the selective pressure.

17. A method for identifying one or more genomic loci associated with a phenotype comprising:

contacting a population of cells with a composition comprising one or more vectors that encode (a) a Cas9 protein and (b) a plurality of CRISPR-Cas system guide RNAs targeting a plurality of different genomic loci, wherein the plurality of CRISPR-Cas system guide RNAs comprise guide RNAs encoded by plasmid library of ATCC Deposit No. PTA121339, guide RNAs encoded by plasmid library of ATCC Deposit No. 121340, guide RNAs encoded by plasmid library of ATCC Deposit No. 121341, guide RNAs encoded by plasmid library of ATCC Deposit No. 121342, guide RNAs encoded by plasmid library of ATCC Deposit No. 121343, or a combination thereof,

selecting cells successfully transduced with the vectors, wherein the selected cells each comprises a CRISPR-Cas system comprising the Cas9 protein and one of the guide RNAs;

applying an selective pressure to the selected cells to obtain cells having a phenotype; and

profiling the depletion or enrichment of the guide RNAs in the cells having the phenotype by high-throughput sequencing to identify one or more genomic loci associated with the phenotype.

Assignments (2)

Continuity (6)

References Cited (400)