Detection of nuclease edited sequences in automated modules and instruments

Patent metadata

Field	Value
Publication number	US-11268061-B2
Application number	US-202117401362-A
Country	US
Kind code	B2
Filing date	Aug 13, 2021
Priority date	Aug 14, 2018
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The present disclosure provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.

First claim

We claim:

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;
a receptacle configured to receive editing nucleic acids;
a growth module for growing cells;
a selection module;
a transformation module configured to introduce the editing nucleic acids into the cells;
a singulation and editing module configured to isolate the transformed cells and allow the editing nucleic acids to edit nucleic acids in the cells wherein the singulation and editing module comprises a device for emulsion formation, comprising: a microfluidic device having an emulsion formation unit including a sample well configured to receive cells in aqueous medium; a carrier fluid well configured to receive a fluid that is immiscible with the cells in aqueous medium; a collection substrate to collect aqueous droplets formed in the immiscible fluid; a sample channel extending from the sample well to a channel intersection; a carrier fluid channel extending from the carrier fluid well to the channel intersection; a droplet channel extending from the channel intersection to the collection substrate; and a pneumatic assembly having a pressure source and a pressure sensor, wherein the pneumatic assembly is configured (a) to apply pressure to the emulsion formation unit to drive generation of droplets at the channel intersection of the emulsion formation unit and collect droplets in the collection substrate, (b) to monitor the pressure with the pressure sensor, and (c) to stop application of the pressure to the emulsion formation unit when the pressure sensor detects a change in pressure indicative of air entering the sample channel from the sample well;
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, from the nucleic acid receptacle to the transformation module, from the transformation module to the selection module and/or from the singulation and editing module; and from the transformation module to the singulation and editing module without user intervention.

2. The automated stand-alone multi-module cell editing instrument of claim 1 , wherein the singulation and editing module further comprises a detection station downstream from the channel intersection but before the collection substrate.

3. The automated stand-alone multi-module cell editing instrument of claim 2 , wherein the detection station comprises a camera.

4. The automated stand-alone multi-module cell editing instrument of claim 2 , further comprising a temperature-controlled editing reservoir positioned between the channel intersection and the detection station.

5. The automated stand-alone multi-module cell editing instrument of claim 4 , wherein the detection station detects the optical density of cells in the aqueous droplets.

6. The automated stand-alone multi-module cell editing instrument of claim 5 , further comprising a droplet sorter positioned between the detection station and the collection substrate.

7. The automated stand-alone multi-module cell editing instrument of claim 6 , wherein the collection substrate comprises two receptacles.

8. The automated stand-alone multi-module cell editing instrument of claim 1 , wherein the collection substrate comprises wells, and is configured to collect one droplet per well.

9. The automated stand-alone multi-module cell editing instrument of claim 8 , wherein the collection substrate is temperature-controlled.

10. The automated stand-alone multi-module cell editing instrument of claim 9 , further comprising a detection station configured to detect droplets in the collection substrate.

11. A method for isolating and editing cells in the automated stand-alone multi-module cell editing instrument of claim 1 , comprising the steps of:
providing live cells in the receptacle configured to receive the live cells;
providing editing nucleic acids the receptacle configured to receive editing nucleic acids;
growing the live cells in a growth module to a desired optical density to produce grown cells;
transforming the grown cells in a transformation module configured to introduce the editing nucleic acids into the filtered cells to produce transformed cells;
selecting for transformed cells to produce selected cells;
generating droplets in the microfluidic device by providing the selected cells in an aqueous medium in the sample well; providing the fluid immiscible with the selected cells in the aqueous medium in the carrier fluid well; flowing the immiscible fluid from the carrier fluid well through the carrier channel to the channel intersection; flowing the selected cells in aqueous medium from the sample well through the sample channel to the channel intersection; generating aqueous droplets in the immiscible fluid; and collecting the aqueous droplets in wells in the collection substrate;
incubating the aqueous droplets in the collection substrate to allow the editing nucleic acids to edit the selected cells;
pooling the aqueous droplets; and
using an automated liquid handling system to 1) transfer the editing nucleic acids from the receptacle 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 cells from the growth module to the transformation module; 4) transfer the transformed cells from the transformation module to the selection module, 5) transfer the selected cells to the sample well, and 6) transfer the cells from the collection substrate to a vessel without user intervention.

12. The method of claim 11 , wherein the fluid immiscible with the cells in the aqueous medium is decane.

13. The method of claim 11 , wherein the generated aqueous droplets comprise cells in a Poisson distribution.

14. The method of claim 11 , wherein the cells are bacterial cells.

15. The method of claim 11 , after the pooling step, filtering the edited cells in a filtration module.

16. A method for isolating and editing cells in the automated stand-alone multi-module cell editing instrument of claim 2 , comprising the steps of:
providing live cells in the receptacle configured to receive the live cells;
providing editing nucleic acids the receptacle configured to receive editing nucleic acids;
growing the live cells in a growth module to a desired optical density to produce grown cells;
transforming the grown cells in a transformation module configured to introduce the editing nucleic acids into the grown cells to produce transformed cells;
selecting the transformed cells in a selection module to produce selected cells;
generating droplets in the microfluidic device by providing the selected cells in an aqueous medium in the sample well; providing the fluid immiscible with the selected cells in the aqueous medium in the carrier fluid well; flowing the immiscible fluid from the carrier fluid well through the carrier channel to the channel intersection; flowing the selected cells in aqueous medium from the sample well through the sample channel to the channel intersection; generating aqueous droplets in the immiscible fluid; and collecting the aqueous droplets one at a time in wells in the collection substrate;
incubating the aqueous droplets in the collection substrate to allow the editing nucleic acids to edit

Assignees

• Inscripta Inc

Inventors

• Garst Andrew
• Graige Michael
• Fox Richard
• Spindler Eileen
• Hiddessen Amy
• Belgrader Phillip
• Masquelier Don
• Chabansky Bruce

Classifications

• C12N15/11

  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 title

• C12N2310/20

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

• C12M35/08 Primary

  Chemical, biochemical or biological means, e.g. plasma jet, co-culture · CPC title

• C12M35/02 Primary

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

• C12M23/44

  Multiple separable units; Modules · CPC title