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

Patent metadata

Field	Value
Publication number	US-10844344-B2
Application number	US-202016820324-A
Country	US
Kind code	B2
Filing date	Mar 16, 2020
Priority date	Aug 14, 2018
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

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 CRISPR editing comprising:
transforming cells with one or more vectors comprising a promoter driving transcription of a coding sequence for a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid sequence covalently-linked to a DNA donor sequence;
diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a substrate;
growing the substantially singulated transformed cells on the substrate until the substantially singulated transformed cells establish colonies;
initiating editing by inducing the inducible promoter driving transcription of the guide nucleic acid; and
growing the edited cells to form colonies of terminal size.

2. The method of claim 1 , wherein the inducible promoter is a pL promoter.

3. The method of claim 1 , wherein all of the coding sequence for the CRISPR nuclease, guide nucleic acid sequence covalently-linked DNA donor sequence are on the same vector.

4. The method of claim 1 , wherein the coding sequence for the CRISPR nuclease is on a first vector and the guide nucleic acid sequence and DNA donor sequence are on a second vector.

5. The method of claim 1 , wherein the DNA donor sequence further comprises a PAM-altering sequence.

6. The method of claim 1 , wherein the edited cells are mammalian cells.

7. The method of claim 1 , wherein the edited cells are yeast cells.

8. The method of claim 1 , wherein the edited cells are bacterial cells.

9. The method of claim 8 , wherein the one or more vectors further comprises a recombineering system.

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

11. The method of claim 10 , wherein the device with solid walls is a solid wall isolation, induction, and normalization (SWIIN) device.

12. A method for enriching edited cells during CRISPR nuclease editing comprising:
transforming cells with one or more vectors comprising a promoter driving transcription of a coding sequence for a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid sequence covalently-linked to a DNA donor sequence, and a gene for a selectable marker;
diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a solid wall device;
growing the substantially singulated transformed cells on the substrate until the substantially singulated transformed cells establish colonies;
initiating editing by inducing the inducible promoter; and
growing the edited cells to form colonies of terminal size.

13. The method of claim 12 , wherein the promoter driving transcription of the guide nucleic acid is a pL promoter.

14. The method of claim 12 , wherein the solid wall device is a solid wall isolation, induction, and normalization (SWIIN) device.

15. The method of claim 12 , wherein the one or more vectors each comprise a selectable marker and further comprising adding selective agents to the substrate to select for the one or more vectors.

16. The method of claim 12 , wherein the CRISPR nuclease is a MADzyme nuclease.

17. The method of claim 12 , further comprising pooling the terminal-size colonies.

18. The method of claim 17 , further comprising subjecting the pooled terminal-size colonies to another round of transformation, dilution, growth, editing, and growth to terminal-size colonies.

19. The method of claim 12 , wherein the transformed cells are mammalian cells.

20. The method of claim 12 , wherein the transformed cells are bacterial cells and the one or more vectors further comprise a recombineering system.

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