Automated cell processing methods, modules, instruments, and systems

• US 10,253,316 B2
• Filed: 06/30/2018
• Issued: 04/09/2019
• Est. Priority Date: 06/30/2017
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Masquelier, Don, Belgrader, Phillip, Bernate, Jorge, Gill, Ryan, Ness, Kevin
• Primary Examiner(s): Bowers, Nathan A
• Application Number:
  US16/024,816
• Publication Number:
  US 20190002814A1
• Time in Patent Office: 283 Days
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 33/14 with filters, sieves or membranes
• CPC Class Codes: C12M 35/00 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 Class Codes: C12M 37/04 Seals
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12M 43/00 Combinations of bioreactors or fermenters with other apparatus
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• 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 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 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

Claims

1. An automated stand-alone multi-module cell editing instrument comprising:

a housing configured to house all or some of the modules;

a receptacle configured to receive cells;

one or more receptacles configured to receive nucleic acids;

a growth module for growing cells for transformation;

a transformation module configured to introduce the nucleic acids into the cells;

a nuclease-directed editing module configured to allow the introduced nucleic acids to edit nucleic acids in the cells;

a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a pre-programmed script; and

an automated liquid handling system to move liquids from the cell receptacle to the growth module, from the growth module to the transformation module and from the transformation module to the nuclease-directed editing module without user intervention.

2. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the nucleic acids in the one or more receptacles comprise a backbone and an editing cassette, and the automated multi-module cell editing instrument further comprises a nucleic acid assembly module.

3. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the automated liquid handling system comprises a sipper or pipettor.

4. The automated stand-alone multi-module cell editing instrument of claim 2, wherein the nucleic acid assembly module is configured to perform isothermal nucleic acid assembly.

5. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the editing module is also a recovery module.

6. The automated stand-alone multi-module cell editing instrument of claim 1, further comprising a recovery module separate from the editing module.

7. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the growth module measures optical density of the growing cells.

8. The automated stand-alone multi-module cell editing instrument of claim 7, wherein the growth module is configured to measure optical density of growing cells continuously.

9. The automated stand-alone multi-module cell editing instrument of claim 7, wherein the processor is configured to adjust growth conditions in the growth module such that the cells reach a target optical density at a time requested by a user.

10. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the receptacle configured to receive cells and the one or more receptacles configured to receive nucleic acids are configured to be contained within a reagent cartridge.

11. The automated stand-alone multi-module cell editing instrument of claim 10, wherein some or all reagents required for cell editing are received by the reagent cartridge.

12. The automated stand-alone multi-module cell editing instrument of claim 11, wherein the reagents contained within the reagent cartridge are locatable by a script read by the processor.

13. The automated stand-alone multi-module cell editing instrument of claim 12, wherein the reagent cartridge includes reagents and is provided in a kit.

14. The automated stand-alone multi-module cell editing instrument of claim 1, wherein the transformation module comprises an electroporation device.

15. The automated stand-alone multi-module cell editing instrument of claim 14, wherein the electroporation device is a flow-through electroporation device.

16. The automated stand-alone multi-module cell editing instrument of claim 1, further comprising a filtration module configured to concentrate the cells and render the cells electrocompetent.

17. An automated stand-alone multi-module cell editing instrument comprising:

a housing configured to house some or all of the modules;

a receptacle configured to receive cells;

at least one receptacle configured to receive nucleic acids for desired genome editing events in the cells;

a nucleic acid assembly module configured to assemble a vector backbone and an editing cassette, wherein the nucleic acid assembly module is configured to accept and assemble nucleic acids to facilitate the desired genome editing events in the cells;

a growth module configured to grow the cells;

a transformation module comprising an electroporator to introduce assembled nucleic acids into the cells;

a nuclease-directed editing module configured to allow the assembled nucleic acids to edit nucleic acids in the cells;

a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a script; and

an automated liquid handling system to move liquids from the cell receptacle to the growth module, from the growth module to the transformation module, and from the transformation module to the nuclease-directed editing module, as well as from the nucleic acid receptacle to the nucleic acid assembly module, and from the nucleic acid assembly module to the transformation module, all without user intervention.

18. The automated stand-alone multi-module cell editing instrument of claim 17, further comprising at least one reagent cartridge containing reagents to perform cell editing in the automated multi-module cell editing instrument.

19. The automated stand-alone multi-module cell editing instrument of claim 18, wherein the receptacles for the cells and nucleic acids are disposed within the reagent cartridge.

20. An automated stand-alone multi-module cell editing instrument comprising:

a housing configured to house some or all of the modules;

a growth module configured to grow cells;

a filtration module configured to concentrate the cells and render the cells electrocompetent;

a transformation module comprising a flow-through electroporator to introduce assembled nucleic acids into the cells;

a combination recovery and nuclease-directed editing module configured to allow the cells to recover after electroporation in the transformation module and to allow the nucleic acids to edit the cells;

a processor configured to operate the automated multi-module cell editing instrument based on user input; and

an automated liquid handling system to move liquids from the growth module to the filtration module, from the filtration module to the transformation module, and from the transformation module to the recovery and nuclease-directed editing module, without user intervention.