Instruments, modules, and methods for improved detection of edited sequences in live cells

Abstract

The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

1 . A module for an automated multi-module cell editing instrument comprising:
 a retentate serpentine port for receiving a population of cells;   a first retentate serpentine channel comprising an upper surface configured to receive the population of cells and a lower surface configured to dispense the received population of cells;   a first permeate serpentine channel disposed under the first retentate serpentine configured to receive the dispensed population of cells and distribute the population of cells into a first plurality of perforations;   a first perforated member comprising the first plurality of perforations disposed under and adjacent to the first permeate channel ;   a partition; and   a second retentate serpentine channel disposed under the partition comprising an upper surface configured to receive the population of cells and a lower surface configured to dispense the received population of cells;   a second permeate serpentine channel disposed under the second retentate serpentine configured to receive the dispensed population of cells and distribute the population of cells into a second plurality of perforations;   a second perforated member comprising a second plurality of perforations disposed under and adjacent to the second permeate serpentine channel.   
     
     
         2 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the first or the second plurality of perforations are approximately 150 μm-200 μm in diameter. 
     
     
         3 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the first or the second plurality of perforations comprise approximately 200,000 perforations. 
     
     
         4 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the first or the second plurality of perforations disposed under and adjacent to the permeate serpentine channel are approximately 125 deep. 
     
     
         5 . The module for the automated multi-module cell editing instrument of  claim 1 , further comprising:
 a second partition; and   a third retentate serpentine channel disposed under the second partition comprising an upper surface configured to receive the population of cells and a lower surface configured to dispense the received population of cells;   a third permeate serpentine channel disposed under the third retentate serpentine configured to receive the dispensed population of cells and distribute the population of cells into a second plurality of perforations;   a third perforated member comprising a third plurality of perforations disposed under and adjacent to the third permeate serpentine channel.   
     
     
         6 . The module for the automated multi-module cell editing instrument of  claim 5 , wherein the third plurality of perforations are approximately 150 μm-200 μm in diameter. 
     
     
         7 . The module for the automated multi-module cell editing instrument of  claim 5 , wherein the third plurality of perforations comprise approximately 200,000 perforations. 
     
     
         8 . The module for the automated multi-module cell editing instrument of  claim 5 , wherein the third plurality of perforations disposed under and adjacent to the permeate serpentine channel are approximately 125 deep. 
     
     
         9 . The module for the automated multi-module cell editing instrument of  claim 5 , further comprising a filter with an upper surface and a lower surface, wherein the upper surface of the filter is positioned beneath and adjacent to the lower surface of the third perforated member. 
     
     
         10 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module has a total of approximately 200,000 microwells to 500,000 microwells. 
     
     
         11 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the volume of the microwells is from lnl to 25 nl. 
     
     
         12 . The module for the automated multi-module cell editing instrument of  claim 11 , wherein the volume of the microwells is from lnl to 10 nl. 
     
     
         13 . The module for the automated multi-module cell editing instrument of  claim 12 , wherein the volume of the microwells is from lnl to 4 nl. 
     
     
         14 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module is configured to receive a population of bacterial cells. 
     
     
         15 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module is configured to receive a population of mammalian cells. 
     
     
         16 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module is configured to receive a population of yeast cells. 
     
     
         17 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module is configured to receive a population of archaea cells. 
     
     
         18 . The module for the automated multi-module cell editing instrument of  claim 1 , wherein the module is reusable. 
     
     
         19 . The module for the automated multi-module cell editing instrument of  claim 1 , further comprising a reservoir assembly comprising a reservoir fluidically-coupled to a reservoir access aperture into which fluids or cells or both flow from outside the module into the reservoir; wherein the reservoir assembly is fluidically-coupled to a reservoir channel port from which fluids or cells or both flow from the reservoir into the retentate serpentine ports; and wherein the reservoir assembly is pneumatically-coupled via a first pneumatic access aperture to a pressure source. 
     
     
         20 . The module for the automated multi-module cell editing instrument of  claim 19 , further comprising a second reservoir assembly comprising a second reservoir fluidically-coupled to a second reservoir access aperture into which fluids or cells or both flow from outside the module into the second reservoir; wherein the second reservoir assembly is fluidically-coupled to a second reservoir channel port from which fluids or cells or both flow from the second reservoir into the retentate serpentine ports; and wherein the second reservoir assembly is pneumatically-coupled via a first pneumatic access aperture to a second pressure source.