Automated cell processing methods, modules, instruments, and systems

• US 10,465,185 B1
• Filed: 05/28/2019
• Issued: 11/05/2019
• 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:
  US16/423,289
• Publication Number:
  US 20190316120A1
• Time in Patent Office: 161 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 of creating a cell library of at least 5,000 edited cells using an automated stand-alone multi-module cell editing instrument for nuclease-directed genome editing, comprising the steps of:

providing live cells in a receptacle configured to receive cells;

providing a library of rationally-designed nucleic acids comprising sequences to facilitate nuclease-directed genome edits in the cells in a receptacle configured to receive nucleic acids;

growing the live cells in a growth module;

transforming the live cells in a transformation module with the library of rationally-designed nucleic acids;

providing conditions in a nuclease-directed editing module to allow nuclease-directed editing of nucleic acids in the cells; and

using an automated liquid handling system to move liquids from the receptacle configured to receive the cells to the growth module, from the growth module to the transformation module, from the receptacle configured to receive the rationally-designed nucleic acids to the transformation module, and from the transformation module to the nuclease-directed editing module without user intervention, wherein the nuclease-directed genome edits created in the automated stand-alone multi-module cell editing instrument result in a cell library comprising at least 5,000 cells with rationally-designed genome edits.

2. The method of claim 1, wherein the rationally-designed genome edits in the cells create a saturation mutagenesis cell library.

3. The method of claim 1, wherein the rationally-designed genome edits in the cells create a promoter swap cell library.

4. The method of claim 1, wherein the rationally-designed genome edits in the cells create a terminator swap cell library.

5. The method of claim 1, wherein the rationally-designed genome edits in the cells create a SNP swap cell library.

6. The method of claim 1, wherein the rationally-designed genome edits in the cells create a promoter and terminator swap cell library.

7. The method of claim 1, wherein the library comprises at least 100,000 edited cells.

8. The method of claim 7, wherein the library comprises at least 1,000,000 edited cells.

9. The method of claim 1, wherein the nuclease-directed genome editing is CRISPR-directed genome editing.

10. The method of claim 1, wherein the nuclease-directed genome editing is TALEN-directed genome editing.

11. The method of claim 1, wherein the automated liquid handling system moves the cell library comprising individual cells with rationally-designed genome edits from the nuclease-directed editing module to the growth module.

12. A method of creating a cell library of at least 5,000 cells using an automated stand-alone multi-module cell editing instrument for nuclease-directed genome editing, comprising the steps of:

providing a cell receptacle configured to receive cells;

providing a nucleic acid receptacle configured to receive rationally-designed nucleic acids comprising sequences to facilitate nuclease-directed genome editing events in the cells;

filtering the cells in a filtration module configured to render the cells electrocompetent;

transforming the electrocompetent cells in a transformation module configured to introduce the rationally-designed nucleic acids into the cells;

providing conditions in a nuclease-directed editing module to allow nuclease-directed editing of nucleic acids in the cells; and

using an automated liquid handling system to move liquids from the receptacle configured to receive cells to the filtration module, from the filtration module to the transformation module, from the receptacle configured to receive rationally-designed nucleic acids to the transformation module, and from the transformation module to the nuclease-directed editing module without user intervention, wherein the nuclease-directed genome edits created in the automated stand-alone multi-module cell editing instrument result in a cell library comprising at least 5,000 cells with rationally-designed genome edits.

13. The method of claim 12, wherein the rationally-designed genome edits in the cells create a saturation mutagenesis cell library.

14. The method of claim 12, wherein the rationally-designed genome edits in the cells create a promoter swap cell library.

15. The method of claim 12, wherein the rationally-designed genome edits in the cells create a terminator swap cell library.

16. The method of claim 12, wherein the rationally-designed genome edits in the cells create a SNP swap cell library.

17. The method of claim 12, wherein the rationally-designed genome edits in the cells create a promoter and terminator swap cell library.

18. The method of claim 12, wherein the library comprises at least 100,000 edited cells.

19. The method of claim 18, wherein the library comprises at least 1,000,000 edited cells.

20. The method of claim 12, wherein the nuclease-directed genome editing is CRISPR-directed genome editing.