Automated cell processing methods, modules, instruments, and systems

• US 10,584,334 B1
• Filed: 11/12/2019
• Issued: 03/10/2020
• 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:
  US16/680,643
• Publication Number:
  US 20200080078A1
• Time in Patent Office: 119 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. A method for simultaneously editing up to 10,000,000 genomic sites in a population of live cells in an automated stand-alone multi-module cell editing instrument, comprising the steps of:

providing a population of live cells in a receptacle configured to receive the live cells;

providing first nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive first nucleic acids;

growing the live cells in a growth module to a desired optical density to produce grown live cells;

filtering and rendering electrocompetent the grown live cells in a filtration module to produce electrocompetent live cells;

transforming the electrocompetent live cells in a transformation module configured to introduce the first nucleic acids into the electrocompetent live cells to produce transformed cells;

providing conditions in a nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the transformed cells to produce edited cells; and

using an automated liquid handling system to

     1) transfer the first nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the live cells from the receptacle configured to receive the live cells to the growth module,

     3) transfer the grown live cells from the growth module to the filtration module,

     4) transfer the electrocompetent cells from the filtration module to the transformation module, and

     5) transfer the transformed cells from the transformation module to the nuclease-directed editing module without user intervention.

2. The method of editing live cells according to claim 1, wherein the cells are prokaryotic cells.

3. The method of editing live cells according to claim 2, wherein the cells are gram positive bacterial cells.

4. The method of editing live cells according to claim 2, wherein the cells are gram negative bacterial cells.

5. The method of editing live cells according to claim 1, wherein the cells are archaeal cells.

6. The method of editing live cells according to claim 1, wherein the cells are eukaryotic cells.

7. The method of editing live cells according to claim 6, wherein the cells are animal cells.

8. The method of editing live cells according to claim 7, wherein the cells are amphibian cells.

9. The method of editing live cells according to claim 7, wherein the cells are mammalian cells.

10. The method of editing live cells according to claim 1, further comprising the steps of

providing second nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive second nucleic acids;

transferring the edited cells from the nuclease-directed editing module to the growth module;

growing the edited cells in the growth module to a desired optical density to produce grown edited cells;

filtering and rendering electrocompetent the grown edited cells in a filtration module to produce electrocompetent edited cells;

transforming the electrocompetent edited cells in the transformation module configured to introduce the second nucleic acids into the electrocompetent edited cells to produce transformed edited cells;

providing conditions in the nuclease-directed editing module to allow the second nucleic acids to edit nucleic acids in the transformed edited cells to produce twice-edited cells; and

using an automated liquid handling system to

     1) transfer the second nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown edited cells from the growth module to the filtration module,

     4) transfer the electrocompetent edited cells from the filtration module to the transformation module, and

     5) transfer the transformed edited cells from the transformation module to the nuclease-directed editing module without user intervention.

11. The method of editing live cells according to claim 10, further comprising the steps of

providing third nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive third nucleic acids;

transferring the twice-edited cells from the nuclease-directed editing module to the growth module;

growing the twice-edited cells in the growth module to a desired optical density to produce grown twice-edited cells;

filtering and rendering electrocompetent the grown twice-edited cells in a filtration module to produce electrocompetent twice-edited cells;

transforming the electrocompetent twice-edited cells in the transformation module configured to introduce the third nucleic acids into the electrocompetent twice-edited cells to produce transformed twice-edited cells;

providing conditions in the nuclease-directed editing module to allow the third nucleic acids to edit nucleic acids in the transformed twice-edited cells to produce thrice-edited cells; and

using an automated liquid handling system to

     1) transfer the third nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the twice-edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown twice-edited cells from the growth module to the filtration module,

     4) transfer the twice-edited electrocompetent cells from the filtration module to the transformation module, and

     5) transfer the transformed twice-edited cells from the transformation module to the nuclease-directed editing module without user intervention.

12. A method for simultaneously editing up to 10,000,000 genomic sites in a population of live cells in an automated stand-alone multi-module cell editing instrument, comprising the steps of:

providing a population of live cells in a receptacle configured to receive the live cells;

providing first nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive first nucleic acids;

growing the live cells in a growth module to a desired optical density to produce grown live cells;

transforming the grown live cells in a transformation module configured to introduce the first nucleic acids into the grown live cells to produce transformed cells;

selecting the transformed cells in a selection module to produce selected cells;

providing conditions in a nuclease-directed editing module to allow the first nucleic acids to edit nucleic acids in the selected cells to produce edited cells; and

using an automated liquid handling system to

     1) transfer the first nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the live cells from the receptacle configured to receive the live cells to the growth module,

     3) transfer the grown live cells from the growth module to the transformation module,

     4) transfer the transformed cells from the transformation module to the selection module, and

     5) transfer the selected cells from the selection module to the nuclease-directed editing module without user intervention.

13. The method of editing live cells according to claim 12, wherein the cells are prokaryotic cells.

14. The method of editing live cells according to claim 12, wherein the cells are eukaryotic cells.

15. The method of editing live cells according to claim 14, wherein the cells are yeast cells.

16. The method of editing live cells according to claim 14, wherein the cells are mammalian cells.

17. The method of editing live cells according to claim 12, further comprising the steps of

providing second nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive second nucleic acids;

transferring the edited cells from the nuclease-directed editing module to the growth module;

growing the edited cells in the growth module to a desired optical density to produce grown edited cells;

transforming the grown edited cells in the transformation module configured to introduce the second nucleic acids into the grown edited cells to produce transformed edited cells;

selecting the transformed edited cells in the selection module to produce selected edited cells;

providing conditions in the nuclease-directed editing module to allow the second nucleic acids to edit nucleic acids in the selected edited cells to produce twice-edited cells; and

using an automated liquid handling system to

     1) transfer the second nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown edited cells from the growth module to the transformation module,

     4) transfer the transformed edited cells from the transformation module to the selection module, and

     5) transfer the selected edited cells from the selection module to the nuclease-directed editing module without user intervention.

18. The method of editing live cells according to claim 17, further comprising the steps of

providing third nucleic acids comprising up to 10,000,000 editing cassettes to one or more receptacles configured to receive third nucleic acids;

transferring the twice-edited cells from the nuclease-directed editing module to the growth module;

growing the twice-edited cells in the growth module to a desired optical density to produce grown twice-edited cells;

transforming the grown twice-edited cells in the transformation module configured to introduce the third nucleic acids into the grown twice-edited cells to produce transformed twice-edited cells;

selecting the transformed twice-edited cells in the selection module to produce selected twice-edited cells;

providing conditions in the nuclease-directed editing module to allow the third nucleic acids to edit nucleic acids in the selected twice-edited cells to produce thrice-edited cells; and

using an automated liquid handling system to

     1) transfer the third nucleic acids from the one or more receptacles configured to receive nucleic acids to the transformation module,

     2) transfer the twice-edited cells from the nuclease-directed editing module to the growth module,

     3) transfer the grown twice-edited cells from the growth module to the transformation module,

     4) transfer the twice-edited transformed cells from the transformation module to the selection module, and

     5) transfer the selected twice-edited cells from the selection module to the nuclease-directed editing module without user intervention.

19. The method of editing live cells according to claim 12, wherein the selection module and the nuclease-directed editing module are a same module.

20. The method of editing live cells according to claim 12, wherein the selection module and the nuclease-directed editing module are separate modules.