Instruments, modules, and methods for improved detection of edited sequences in live cells

Patent metadata

Field	Value
Publication number	US-11046928-B2
Application number	US-202017102414-A
Country	US
Kind code	B2
Filing date	Nov 23, 2020
Priority date	Aug 14, 2018
Publication date	Jun 29, 2021
Grant date	Jun 29, 2021

Abstract

The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

First claim

We claim:

1. A method for enriching edited cells during nucleic acid-guided CRISPR nuclease editing comprising:
diluting a population of cells into groups of 2 to 100 cells thereby generating substantially singulated groups of cells, wherein each cell comprises a promoter driving expression of a coding sequence for a nucleic acid-guided nuclease and a promoter driving expression of a guide nucleic acid;
growing the cells in the substantially singulated groups of cells;
initiating editing of the cells in the substantially singulated groups of cells by inducing transcription of one or both of the nucleic acid-guided nuclease and the guide nucleic acid;
growing the edited cells in the substantially singulated groups of cells on a substrate until the edited cells in the substantially singulated groups of cells establish colonies of terminal size.

2. The method of claim 1 , wherein the same inducible promoter drives transcription of both of the gRNA and the nuclease.

3. The method of claim 1 , wherein the population of cells is diluted into groups of 2 to 50 cells.

4. The method of claim 1 , wherein the population of cells is diluted into groups of 2 to 10 cells.

5. The method of claim 1 , wherein the inducing transcription comprises inducing one or more temperature inducible promoter.

6. The method of claim 1 , wherein the inducing transcription comprises inducing one or more pBAD promoter(s).

7. The method of claim 1 , wherein cells in the substantially singulated groups of cells further comprise a nucleic acid sequence for a donor nucleic acid.

8. The method of claim 7 , wherein the donor nucleic acid is covalently linked to the guide nucleic acid.

9. The method of claim 8 , wherein the device with solid walls is a solid wall isolation, induction, and normalization (SWIIN) device.

10. The method of claim 7 , wherein the nucleic acid sequence for the donor nucleic acid encodes a promoter sequence for a promoter swap.

11. The method of claim 1 , wherein the edited cells are bacterial cells, mammalian cells, or yeast cells.

12. The method of claim 1 , wherein the substrate is selected from an agar plate and a device with solid walls.

13. A method for enriching edited cells during nucleic acid-guided CRISPR nuclease editing comprising:
diluting a populations of cells into groups of 2 to 100 cells thereby generating substantially singulated groups of cells, wherein the cells comprise a promoter driving expression of a coding sequence for a nucleic acid-guided nuclease and a promoter driving expression of a guide nucleic acid;
growing the cells in the substantially singulated groups of cells on a substrate;
initiating editing of cells in the substantially singulated groups of cells by inducing transcription of one or both of the nucleic acid-guided nuclease and the guide nucleic acid;
growing the substantially singulated groups of cells on the substrate then selecting slow-growing colonies.

14. The method of claim 13 , wherein the same inducible promoter drives transcription of both of the gRNA and the nuclease.

15. The method of claim 13 , wherein the population of cells is diluted into groups of 2 to 50 cells.

16. The method of claim 13 , wherein the population of cells is diluted into groups of 2 to 10 cells.

17. The method of claim 13 , wherein the inducing transcription comprises inducing one or more temperature inducible promoter.

18. The method of claim 13 , wherein cells in the substantially singulated groups of cells further comprise a nucleic acid sequence for a donor nucleic acid.

19. The method of claim 18 , wherein the nucleic acid sequence for the donor nucleic acid encodes a promoter sequence for a promoter swap.

20. The method of claim 13 , wherein the edited cells are bacterial cells, mammalian cells, or yeast (Original) cells.

Assignees

• Inscripta Inc

Inventors

• Garst Andrew
• Fox Richard
• Belgrader Phillip
• Masquelier Don

Classifications

• C12N2800/80

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

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/22 Primary

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

• C12M47/04 Primary

  Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04) · CPC title