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

• US 10,533,152 B1
• Filed: 04/30/2019
• Issued: 01/14/2020
• Est. Priority Date: 08/14/2018
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Belgrader, Phillip, Masquelier, Don, Chabansky, Bruce, Bernate, Jorge, Garst, Andrew, Fox, Richard
• Primary Examiner(s): Patel, Pranav N
• Application Number:
  US16/399,988
• Time in Patent Office: 259 Days
• CPC Class Codes: B01D 2201/0407 Perforated supports on both sides of the filtering element
• CPC Class Codes: B01D 2313/50 Specific extra tanks
• CPC Class Codes: B01D 2315/10 Cross-flow filtration
• CPC Class Codes: B01D 63/082 comprising a stack of flat membranes
• CPC Class Codes: B01D 69/06 Flat membranes
• CPC Class Codes: B01D 71/0213 Silicon
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/68 Stabilisation of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2830/001 : controllable enhancer/promoter combination
• CPC Class Codes: C12N 2830/002 : inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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 singulation or substantial singulation, growth, induction of editing, and normalization or cherry-picking (SWIIN) module comprising:

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 retentate ridges between which are retentate flow directors;

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;

a perforated member with an upper surface and a lower surface, wherein the upper surface of the perforated member is positioned beneath and adjacent to the lower surface of the retentate member and wherein the perforated member comprises at least 25,000 perforations;

a filter with an upper surface and a lower surface, wherein the upper surface of the filter is positioned beneath and adjacent to the lower surface of the perforated member and wherein the lower surface of the filter is positioned above and adjacent to an upper surface of a permeate member;

a gasket surrounding the perforated member and the filter; and

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 permeate ridges between which are permeate flow directors;

wherein the permeate member further comprises one or more permeate member ports configured to supply fluid to and remove fluid from the permeate member; and

wherein the permeate member ports are fluidically-connected to the at least one permeate distribution channel and permeate flow directors; and

means to couple the retentate member, perforated member, filter, gasket and permeate member.

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

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 SWIIN module of claim 2, further comprising:

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 singulation assembly of claim 1, wherein the means to couple the retentate member, perforated member, filter, gasket and permeate member comprises ultrasonic welding.

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

6. The singulation assembly of claim 5, wherein the perforated member comprises at least 200,000 perforations.

7. The singulation assembly of claim 1, wherein the retentate and permeate members are fabricated from polycarbonate, cyclic olefin co-polymer, or poly(methyl methylacrylate).

8. The singulation assembly of claim 1, wherein the retentate and permeate members 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.

9. The singulation assembly of claim 8, wherein the retentate and permeate members are 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.

10. The singulation assembly of claim 1, wherein there are two retentate distribution channels.

11. The singulation assembly of claim 10, wherein the two retentate distribution channels are approximately 150 mm in length and 1 mm in width.

12. The singulation assembly of claim 1, wherein there are two permeate distribution channels.

13. The singulation assembly of claim 12, wherein the two permeate distribution channels are approximately 150 mm in length and 1 mm in width.

14. The singulation assembly of claim 1, wherein the retentate ridges are approximately 0.5 mm in height and 80 mm in length.

15. The singulation assembly of claim 1, wherein the retentate flow directors are approximately 5 mm across.

16. The singulation assembly of claim 1, wherein the permeate ridges are approximately 0.5 mm in height and 80 mm in length.

17. The singulation assembly of claim 1, wherein the permeate flow directors are approximately 5 mm across.

18. The singulation assembly of claim 1, wherein a volume of the singulation assembly is from 15 mL to 40 mL.

19. A SWIIN module comprising:

a singulation assembly comprising;

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 retentate ridges between which are retentate flow directors;

wherein the one or more 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;

a perforated member with an upper surface and a lower surface, wherein the upper surface of the perforated member is positioned beneath and adjacent to the lower surface of the retentate member;

a filter with an upper surface and a lower surface, wherein the upper surface of the filter is positioned beneath and adjacent to the lower surface of the perforated member and wherein the lower surface of the filter is positioned above and adjacent to an upper surface of a permeate member;

a gasket surrounding the perforated member and the filter; and

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 permeate ridges between which are permeate flow directors;

wherein the permeate member further comprises one or more permeate member ports configured to supply fluid to and remove fluid from the permeate member; and

wherein the one or more permeate member ports are fluidically-connected to the at least one permeate distribution channel and permeate flow directors; and

means to couple the retentate member, perforated member, filter, gasket and permeate member;

a reservoir assembly comprising at least two reservoirs wherein

a first reservoir is

     1) fluidically-coupled to a first reservoir aperture into which fluids and/or cells flow from outside the SWIIN module into the first reservoir,

     2) fluidically-coupled to at least one 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 a second reservoir access aperture into which fluids and/or cells 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.