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

• US 10,625,212 B2
• Filed: 11/18/2019
• Issued: 04/21/2020
• Est. Priority Date: 08/14/2018
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Masquelier, Don, Belgrader, Phillip, Garst, Andrew, Fox, Richard, Estes, Matthew, Chabansky, Bruce, Bernate, Jorge
• Primary Examiner(s): Patel, Pranav N
• Application Number:
  US16/687,640
• Publication Number:
  US 20200078738A1
• Time in Patent Office: 155 Days
• CPC Class Codes: B01D 2201/0407 Perforated supports on both sides of the filtering element
• CPC Class Codes: B01D 2313/041 Gaskets or O-rings
• CPC Class Codes: B01D 2313/042 Adhesives or glues
• CPC Class Codes: B01D 2313/08 Flow guidance means within the module or the apparatus
• CPC Class Codes: B01D 2313/086 Meandering flow path over the membrane
• CPC Class Codes: B01D 2313/105 Supply manifolds
• CPC Class Codes: B01D 2313/20 Specific housing
• CPC Class Codes: B01D 2313/205 characterised by the shape
• CPC Class Codes: B01D 2313/2061 Organic, e.g. polymeric material
• CPC Class Codes: B01D 2313/50 Specific extra tanks
• CPC Class Codes: B01D 25/003 integrally combined with devices for controlling the filtration
• CPC Class Codes: B01D 61/18 Apparatus therefor
• CPC Class Codes: B01D 63/087 Single membrane modules
• CPC Class Codes: B01D 63/088 Microfluidic devices comprising semi-permeable flat membranes
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12N 15/00 : Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/1003 : Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/102 : Mutagenizing nucleic acids

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.

Claims

1. A singulation assembly for a solid wall isolation, incubation and normalization (SWIIN) module comprising:

a retentate member comprising;

an upper surface and a lower surface and a first and second end,

an upper portion of a serpentine channel defined by raised areas on the lower surface of the retentate member, wherein the upper portion of the serpentine channel traverses the lower surface of the retentate member for about 50% to about 90% of a length and width of the lower surface of the retentate member; and

at least one retentate port fluidically connected to the serpentine channel; and

a permeate member disposed under the retentate member comprising;

an upper surface and a lower surface and a first and second end,

a lower portion of the serpentine channel defined by raised areas on the upper surface of the permeate member, wherein the lower portion of the serpentine channel traverses the upper surface of the permeate member for about 50% to about 90% of a length and width of the upper surface of the permeate member, and wherein the lower portion of the serpentine channel is configured to mate with the upper portion of the serpentine channel to form the serpentine channel;

at least one permeate port fluidically connected to the serpentine channel;

a perforated member comprising at least 25,000 perforations disposed under and adjacent to the retentate member;

a filter disposed under and adjacent to the perforated member and above and adjacent to the permeate member; and

a gasket surrounding the filter.

2. A SWIIN module comprising the singulation assembly of claim 1 further comprising:

first and second reservoirs at the first end of the permeate member, wherein the first reservoir is fluidically connected to the at least one permeate port in the permeate member and the second reservoir is fluidically connected to the at least one retentate port in the retentate member;

a reservoir cover at the first end of the retentate member configured to cover the first and second reservoirs; and

a gasket disposed on top of the reservoir cover, wherein the gasket comprises for each of the first and second reservoirs a reservoir access aperture configured to provide fluid access to a reservoir and a pneumatic access aperture configured to provide pneumatic access to a reservoir.

3. The SWIIN module of claim 2, wherein the reservoir access apertures receive fluid from outside the SWIIN module or remove fluid from the reservoirs.

4. The SWIIN module of claim 2, further comprising a third and a fourth reservoir wherein the third reservoir is 1) fluidically coupled to a second port in the retentate member, 2) fluidically coupled to a third reservoir access aperture in the gasket disposed on top of the reservoir cover into which fluids and/or cells flow from outside the SWIIN module into the third reservoir, and 3) pneumatically coupled to a pressure source;

and wherein the fourth reservoir is

     1) fluidically coupled to a second port in the permeate member,

     2) fluidically coupled to a fourth reservoir access aperture in the gasket on top of the reservoir cover into which fluids and/or cells flow from outside the SWIIN module into the fourth reservoir, and

     3) pneumatically coupled to a pressure source.

5. The singulation assembly of claim 1, wherein the permeate member further comprises ultrasonic tabs disposed on the raised areas on the upper surface of the permeate member and at the first and second end of the permeate member;

the retentate member further comprises recesses for the ultrasonic tabs, wherein the recesses are disposed on the raised areas on the lower surface of the retentate member and at the first and second end of the retentate member;

the ultrasonic tabs are configured to mate with the recesses for the ultrasonic tabs; and

the permeate member, the retentate member, the perforated member and the filter are coupled together by ultrasonic welding.

6. The singulation assembly of claim 1, wherein the permeate member, the retentate member, the perforated member and the filter are coupled together by a pressure sensitive adhesive.

7. The singulation assembly of claim 1, wherein the perforated member comprises at least 100,000 perforations.

8. The singulation assembly of claim 1, wherein the perforated member comprises at least 200,000 perforations.

9. The singulation assembly of claim 1, wherein the perforated member comprises at least 400,000 perforations.

10. The singulation assembly of claim 1, wherein the retentate member is fabricated from polycarbonate, cyclic olefin co-polymer, or poly(methyl methylacrylate).

11. The singulation assembly of claim 1, wherein a serpentine channel portion of each of the retentate and permeate members is 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.

12. The singulation assembly of claim 1, wherein a serpentine channel portion of each of the retentate and permeate members is from 150 mm to 250 mm in length, from 100 mm to 150 mm in width, and from 4 mm to 8 mm in thickness.

13. The singulation assembly of claim 1, wherein the volume of the mated serpentine channel is from 4 to 40 mL.

14. The singulation assembly of claim 1, wherein the volume of a well formed by one of the perforations is from 1 to 5 nL.

15. A SWIIN module comprising the singulation assembly of claim 1, wherein the SWIIN module further comprises imaging means to detect cells growing in wells.

16. The SWIIN module of claim 15, wherein the imaging means comprises a camera and a backlight positioned beneath the permeate member.

17. The SWIIN module of claim 15, further comprising a thermoelectric control device, and a fan.

18. A SWIIN module comprising:

a singulation assembly comprising;

a retentate member comprising;

an upper surface and a lower surface and a first and second end,

an upper portion of a serpentine channel defined by raised areas on the lower surface of the retentate member, wherein the upper portion of the serpentine channel traverses the lower surface of the retentate member for about 50% to about 90% of a length and width of the lower surface of the retentate member; and

at least one retentate port fluidically connected to the upper portion of the serpentine channel; and

a permeate member disposed under the retentate member comprising;

an upper surface and a lower surface and a first and second end,

a lower portion of the serpentine channel defined by raised areas on the upper surface of the permeate member, wherein the lower portion of the serpentine channel traverses the upper surface of the permeate member for about 50% to about 90% of a length and width of the upper surface of the permeate member, and wherein the lower portion of the serpentine channel is configured to mate with the upper portion of the serpentine channel to form the serpentine channel;

at least one permeate port fluidically connected to the lower portion of the serpentine channel;

a perforated member comprising at least 25,000 perforations disposed under and adjacent to the retentate member;

a filter disposed under and adjacent to the perforated member and above and adjacent to the permeate member; and

a gasket surrounding the filter; and

first and second reservoirs at the first end of the permeate member, wherein the first reservoir is fluidically connected to the at least one permeate port in the permeate member and the second reservoir is fluidically connected to the at least one retentate port in the retentate member;

a reservoir cover at the first end of the retentate member; and

a gasket disposed on top of the reservoir cover, wherein the gasket comprises for each of the first and second reservoir a reservoir access aperture configured to provide fluid access to a reservoir and a pneumatic access aperture configured to provide pneumatic access to a reservoir.

19. The SWIIN module of claim 18, further comprising a third and a fourth reservoir wherein the third reservoir is 1) fluidically coupled to a second port in the retentate member, 2) fluidically coupled to a third reservoir access aperture in the gasket on top of the reservoir cover into which fluids and/or cells flow from outside the SWIIN module into the third reservoir, and 3) pneumatically coupled to a pressure source;

and wherein the fourth reservoir is

     1) fluidically coupled to a second port in the permeate member,

     2) fluidically coupled to a fourth reservoir access aperture in the gasket on top of the reservoir cover into which fluids and/or cells flow from outside the SWIIN module into the fourth reservoir, and

     3) pneumatically coupled to a pressure source.

20. The SWIIN module of claim 18, wherein the reservoir access apertures receive fluid from outside the SWIIN module or remove fluid from the reservoirs.