Automated cell processing methods, modules, instruments, and systems

Patent metadata

Field	Value
Publication number	US-11203751-B2
Application number	US-202117336244-A
Country	US
Kind code	B2
Filing date	Jun 1, 2021
Priority date	Jun 30, 2017
Publication date	Dec 21, 2021
Grant date	Dec 21, 2021

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.

First claim

We claim:

1. A method of editing mammalian cells in an automated stand-alone multi-module cell editing instrument comprising:
providing 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 the mammalian cells;
one or more receptacles configured to receive expression vectors, wherein the expression vectors introduce a desired DNA change to the target region and remove a proto-spacer motif (PAM) region from the target region;
a growth module for growing cells for transformation;
a transformation module configured to introduce the expression vectors into the mammalian cells;
a nuclease-directed editing module configured to allow the introduced expression vectors to edit nucleic acids in the mammalian 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 receptacle configured to receive the mammalian cells to the growth module; from the growth module to the transformation module; from the one or more receptacles configured to receive expression vectors to the transformation module; and from the transformation module to the nuclease-directed editing module without user intervention;

loading mammalian cells into the receptacle configured to receive the mammalian cells;
loading the expression vectors into the one or more receptacles configured to receive expression vectors;
transferring, via the liquid handling system, the mammalian cells from the receptacle configured to receive the mammalian cells to the growth module;
growing the mammalian cells;
transferring, via the liquid handling system, the grown mammalian cells from the growth module to the transformation module;
transferring, via the liquid handling system, the expression vectors from the one or more receptacles to the transformation module;
transforming the grown mammalian cells with the expression vectors to produce transformed mammalian cells;
transferring, via the liquid handling system, the transformed mammalian cells from the transformation module into the nuclease-directed editing module; and
editing the transformed mammalian cells to produce edited mammalian cells.

2. The method of editing mammalian cells of claim 1 , wherein the automated liquid handling system comprises a sipper or pipettor.

3. The method of editing mammalian cells of claim 1 , wherein the nuclease-directed editing module is also a recovery module.

4. The method of editing mammalian cells of claim 1 , wherein the automated stand-alone multi-module cell editing instrument further comprises a recovery module separate from the editing module and further comprising after the editing step, the step of transferring the edited mammalian cells from the nuclease-directed editing module to the recovery module.

5. The method of editing mammalian cells of claim 1 , wherein the growth module is separate from the transformation module.

6. The method of editing mammalian cells of claim 1 , wherein the transformation module performs a process selected from electroporation, lipofection, optoporation, injection, microprecipitation, magnetofection, microinjection, particle bombardment, sonoporation, laser-induced poration, bead transfection, calcium phosphate and calcium chloride co-precipitation, and DEAE-dextran-mediated transfection.

7. The method of editing mammalian cells of claim 6 , wherein the transformation module performs electroporation.

8. The method of editing mammalian cells of claim 1 , wherein the automated stand-alone multi-module cell editing instrument further comprises a selection module.

9. The method of editing mammalian cells of claim 1 , wherein the growth module and the transformation module are combined.

10. The method of editing mammalian cells of claim 1 , wherein the growth module is combined with the transformation module.

11. The method of editing mammalian cells of claim 1 , wherein the growth module, transformation module and nuclease-directed editing module are combined.

12. A method of editing mammalian cells in an automated stand-alone multi-module cell editing instrument comprising:
providing 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 the mammalian cells;
one or more receptacles configured to receive expression vectors, wherein the expression vectors introduce a desired DNA change to the target region and remove a proto-spacer motif (PAM) region from the target region;
a transformation module configured to introduce the expression vectors into the mammalian cells;
a nuclease-directed editing module configured to allow the introduced expression vectors to edit nucleic acids in the mammalian cells;
a selection module to select for edited mammalian 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 receptacle configured to receive the mammalian cells to the transformation module; from the one or more receptacles configured to receive expression vectors to the transformation module; from the transformation module to the nuclease-directed editing module; and from the nuclease-directed editing module to the selection module without user intervention;

loading mammalian cells into the receptacle configured to receive the mammalian cells;
loading the expression vectors comprising the vector backbone and the editing cassette into the one or more receptacles configured to receive expression vectors;
transferring, via the liquid handling system, the mammalian cells from the receptacle configured to receive the mammalian cells to the transformation module;
transferring, via the liquid handling system, the expression vectors from the one or more receptacles to the transformation module;
transforming the mammalian cells with the expression vectors to produce transformed mammalian cells;
transferring, via the liquid handling system, the transformed mammalian cells from the transformation module into the nuclease-directed editing module;
editing the transformed mammalian cells; and
selecting edited mammalian cells.

13. The method of editing mammalian cells of claim 12 , wherein the automated liquid handling system comprises a sipper or pipettor.

14. The method of editing mammalian cells of claim 12 , wherein the growth module is combined with the transformation module.

15. The method of editing mammalian cells of claim 12 , wherein the transformation module, nuclease-directed editing module and selection module are combined.

16. The method of editing mammalian cells of claim 12 , wherein the nuclease-directed editing module is also a recovery module.

17. The method of editing mammalian cells of claim 12 , wherein the transformation module performs a process selected from electroporation, lipofection, optoporation, injection, microprecipitation, magnetofection, microinjection, particle bombardment, sonoporation, laser-induced poration, bead transfection, calcium phosphate and calcium chloride co-precipitation, and DEAE-dextran-mediated transfection.

18. The method of editing mammalian cells of claim 12 , wherein the automated stand-alone multi-module cell editing instrument further comprises a separate growth module a

Assignees

• Inscripta Inc

Inventors

• Bernate Jorge
• Ness Kevin
• Belgrader Phillip
• Masquelier Don
• Gill Ryan

Classifications

• C12M35/02

  Electrical or electromagnetic means, e.g. for electroporation or for cell fusion · CPC title

• C12Q1/6865

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title