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

Patent metadata

Field	Value
Publication number	US-10801008-B1
Application number	US-202016930269-A
Country	US
Kind code	B1
Filing date	Jul 15, 2020
Priority date	Aug 14, 2018
Publication date	Oct 13, 2020
Grant date	Oct 13, 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 creating edited cell libraries comprising:
providing cells to be edited;
providing nucleic acids to edit the cells to be edited;
providing a growth module, a transformation module and a singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWUN) module, wherein the SWIIN module comprises a singulation assembly,
wherein the singulation assembly comprises:
a retentate member comprising an upper surface and a lower surface, wherein the retentate member comprises at least one retentate distribution channel which traverses the retentate member from its upper surface to its lower surface and for 70% to 95% of a length of retentate member; wherein the lower surface of the retentate member comprises a first portion of a serpentine channel; wherein the retentate member further comprises one or more retentate member ports configured to supply cells and fluid to and remove cells and fluid from the retentate member; and wherein the retentate member ports are fluidically-connected to the at least one retentate distribution channel and retentate flow directors;
the permeate member comprising the upper surface and a lower surface, wherein the permeate member comprises at least one permeate distribution channel which traverses the permeate member from its lower surface to its upper surface and for 70% to 95% of a length of permeate member; wherein the upper surface of the permeate member comprises a second portion of the serpentine channel; wherein the permeate member further comprises one or more permeate member ports configured to supply fluid to and remove fluid from the permeate member; wherein the permeate member ports are fluidically-connected to the at least one permeate distribution channel and permeate flow directors; and wherein the first and second portions of the serpentine channel mate to create the serpentine channel;
a gasket disposed between the retentate member and the permeate member; and
means to couple the retentate member and permeate member;

transferring the cells to be edited to the growth module;
growing the cells to be edited;
transferring the grown cells from the growth module to the transformation module;
transferring the nucleic acids to the transformation module;
transforming the grown cells with the nucleic acids;
singulating the transformed cells in the SWIIN module; and
providing conditions in the SWIIN module to edit the cells, thereby creating an edited cell library.

2. The method for creating edited cell libraries of claim 1 , wherein the SWIIN module further comprises:
a reservoir assembly comprising at least two reservoirs wherein
a first reservoir is 1) fluidically-coupled to at least a first reservoir access aperture into which fluids and/or cells flow from outside the SWIIN module into the first reservoir, 2) fluidically-coupled to a reservoir/channel port from which fluids and/or cells flow from the first reservoir into the one or more retentate member ports; and 3) pneumatically-coupled via a first pneumatic access aperture to a pressure source;
a second reservoir is 1) fluidically-coupled to at least a second reservoir access aperture into which fluids flow from outside the SWIIN module into the second reservoir, 2) fluidically-coupled to a reservoir/channel port from which fluids and/or cells flow from the second reservoir into the one or more permeate member ports; and 3) pneumatically-coupled via a second pneumatic access aperture to a pressure source; and

a SWIIN cover.

3. The method for creating edited cell libraries of claim 2 , wherein the SWIIN module further comprises:
two additional reservoirs wherein
a third reservoir is 1) fluidically-coupled to a third reservoir access aperture into which fluids and/or cells flow from outside the SWIIN module into the third reservoir, 2) fluidically-coupled to a reservoir/channel port from which fluids and/or cells flow from the third reservoir into the one or more retentate member ports; and 3) pneumatically-coupled via a third pneumatic access aperture to a pressure source; and
a fourth reservoir is 1) fluidically-coupled to a fourth reservoir access aperture into which fluids and/or cells flow from outside the SWIIN module into the fourth reservoir, 2) fluidically-coupled to a reservoir/channel port from which fluids and/or cells flow from the reservoir into the one or more permeate member ports; and 3) pneumatically-coupled via a fourth pneumatic access aperture to a pressure source.

4. The method for creating edited cell libraries of claim 1 , wherein the means to couple the retentate member and perforated member is a pressure sensitive adhesive.

5. The method for creating edited cell libraries of claim 1 , wherein the retentate and permeate members of the singulation assembly are from 75 mm to 350 mm in length, from 50 mm to 250 mm in width, and from 2 mm to 15 mm in thickness.

6. The method for creating edited cell libraries of claim 1 , wherein a volume of the singulation assembly is from 5 mL to 100 mL.

7. The method for creating edited cell libraries of claim 6 , wherein the volume of the singulation assembly is from 10 mL to 40 mL.

8. The method for creating edited cell libraries of claim 1 , further comprising the steps of providing a cell concentration module and, after growing the cells to be edited in the growth module, transferring the grown cells to the cell concentration module and concentrating the grown cells.

9. The method for creating edited cell libraries of claim 1 , wherein the cell library created is a promoter swap library.

10. The method for creating edited cell libraries of claim 1 , wherein the cell library created is a terminator swap library.

11. The method for creating edited cell libraries of claim 1 , wherein the cell library created is a saturation mutagenesis library.

12. The method for creating edited cell libraries of claim 1 , wherein the cell library created comprises at least 1,000 edited cells.

13. The method for creating edited cell libraries of claim 12 , wherein the cell library created comprises at least 10,000 edited cells.

14. The method for creating edited cell libraries of claim 13 , wherein the cell library created comprises at least 100,000 edited cells.

15. The method for creating edited cell libraries of claim 14 , wherein the cell library created comprises at least 500,000 edited cells.

16. The method for creating edited cell libraries of claim 15 , wherein the cell library created comprises at least 1,000,000 edited cells.

17. The method for creating edited cell libraries of claim 16 , wherein the cell library created comprises at least 5,000,000 edited cells.

18. The method for creating edited cell libraries of claim 1 , wherein a liquid handling device performs the transfer and singulating steps.

19. The method for creating edited cell libraries of claim 18 , wherein the liquid handling device is controlled by a processor.

20. The method for creating edited cell libraries of claim 1 , further comprising the step of providing a reagent cartridge.

Assignees

• Inscripta Inc

Inventors

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

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