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

Patent metadata

Field	Value
Publication number	US-11072774-B2
Application number	US-202117175612-A
Country	US
Kind code	B2
Filing date	Feb 13, 2021
Priority date	Aug 14, 2018
Publication date	Jul 27, 2021
Grant date	Jul 27, 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 compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking module (“solid wall isolation/induction/normalization module” or “SWIIN”) comprising: at least three singulation assemblies, wherein each of the at least three singulation assemblies comprise:
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; 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 the first portion of the serpentine channel;
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; 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 second portion of the serpentine channel and permeate flow directors; and wherein the first and second portions of the serpentine channel mate to create the serpentine channel;
a perforated member disposed between the retentate member and the permeate member;
means to couple the retentate member, perforated member and permeate member;
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.

2. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , 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.

3. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein a pressure sensitive adhesive is used to couple the retentate member, perforated member and permeate member in each of the at least three singulation assemblies.

4. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein solvent bonding is used to couple the retentate member, perforated member and permeate member in each of the at least three singulation assemblies.

5. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein mated fittings are used to couple the retentate member, perforated member and permeate member in each of the at least three singulation assemblies.

6. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein the retentate and permeate members of the at least three singulation assemblies 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.

7. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein a volume of fluid in each of the at least three singulation assemblies is from 5 mL to 100 mL.

8. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 7 , wherein the volume of fluid in each of the at least three singulation assemblies is from 7.5 mL to 60 mL.

9. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 8 , wherein the volume of fluid in each of the at least three singulation assemblies is from 10 mL to 40 mL.

10. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 , wherein each perforated member comprises at least 50,000 perforations.

11. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 10 , wherein each perforated member comprises at least 100,000 perforations.

12. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 11 , wherein each perforated member comprises at least 150,000 perforations.

13. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 12 , wherein each perforated member comprises at least 200,000 perforations.

14. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 13 , wherein each perforated member comprises at least 250,000 perforations.

15. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 1 wherein the at least three singulation assemblies comprise at least four singulation assemblies.

16. The compound singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module of claim 15 wherein the at least three singulation assemblies comprise at least five singulation assemblies.

17. The compound singulation or substantial singulation, growth, induction of editing, a

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