Detection of nuclease edited sequences in automated modules and instruments

Patent metadata

Field	Value
Publication number	US-11965154-B2
Application number	US-201916545097-A
Country	US
Kind code	B2
Filing date	Aug 20, 2019
Priority date	Aug 30, 2018
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

The present disclosure provides modules, instruments and methods to enrich for cells edited via nucleic acid-guided nuclease editing of live cells.

First claim

We claim:

1. A method for performing enrichment of cells edited by a nucleic acid-guided nuclease comprising:
(a) providing transformed cells in growth medium, wherein the cells comprise a coding sequence for a nucleic acid-guided nuclease, a sequence for a donor DNA, and a coding sequence for a gRNA, wherein at least the coding sequence for the gRNA is under the control of an inducible promoter;
(b) allowing the transformed cells to grow until the cells are in stationary phase;
(c) inducing transcription of the coding sequence for the gRNA; and
(d) allowing the cells to edit, wherein an editing efficiency is improved compared to inducing transcription of the coding sequence for the gRNA when the transformed cells are in log phase.

2. The method of claim 1 , wherein the coding sequence for a nucleic acid-guided nuclease, the sequence for a donor DNA, and the coding sequence for the gRNA are provided to the cells on a single vector.

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

4. The method of claim 1 , wherein the inducible promoter comprises a first inducible promoter, and wherein the method further comprises rendering the transformed cells electrocompetent and transforming the cells with one or more nucleic acid-guided editing components under the control of a second inducible promoter after step (b).

5. The method of claim 1 , wherein the inducible promoter is a promoter that is activated upon an increase in temperature.

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

7. The method of claim 5 , wherein transcription is induced by raising the temperature of the cells to 42° C.

8. The method of claim 1 , wherein the inducible promoter is a promoter that is activated upon adding an inducing moiety.

9. A method for performing enrichment of cells edited by a nucleic acid-guided nuclease comprising:
(a) providing transformed cells in growth medium, wherein the cells comprise a first round of nucleic acid-guided editing components, wherein the first round of nucleic acid-guided editing components comprises a first gRNA under the control of a first inducible promoter;
(b) allowing the transformed cells to grow until the cells are in stationary phase;
(c) inducing transcription of the first gRNA;
(d) allowing the cells to edit, wherein an editing efficiency is improved compared to inducing transcription of the first gRNA when the transformed cells are in log phase; and
(e) transforming the cells with a second round of nucleic acid-guided editing components, wherein the second round of nucleic acid-guided editing components comprises a second gRNA under the control of a second inducible promoter.

10. The method of claim 9 , wherein the cells are bacterial cells, yeast cells, or mammalian cells.

11. The method of claim 9 , wherein the first round of nucleic acid-guided editing components are encoded on a single vector.

12. The method of claim 9 , wherein the second round of nucleic acid-guided editing components are encoded on a single vector.

13. The method of claim 9 , wherein the first round of nucleic acid-guided editing components and the second round of nucleic acid-guided editing components are encoded on separate vectors.

14. The method of claim 9 , wherein the first inducible promoter, the second inducible promoter, or both, are promoters activated upon an increase in temperature.

15. The method of claim 9 , wherein the first inducible promoter, the second inducible promoter, or both, are pL promoters.

16. The method of claim 14 , wherein transcription is induced by raising the temperature of the cells to 42° C.

17. The method of claim 9 , wherein the first inducible promoter, the second inducible promoter, or both, are promoters activated upon adding an inducing moiety.

Assignees

• Inscripta Inc

Inventors

• Spindler Eileen
• Wagner Isaac
• Davis Clint
• Swavola Julia
• Belgrader Phillip

Classifications

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title

• B01L3/502

  with fluid transport, e.g. in multi-compartment structures · CPC title

• C12M27/10

  Rotating vessel · CPC title

• C12M29/04

  Filters; Permeable or porous membranes or plates, e.g. dialysis · CPC title

• C12M35/00

  Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87) · CPC title