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.

We claim: 
     
         1 . An automated multi-module cell editing instrument comprising:
 a singulation assembly for singulating cells from a population of cells comprising;
 a retentate member comprising an upper surface configured to receive the population of cells and a lower surface configured to dispense the received population of cells; 
 a permeate member disposed under the retentate member configured to receive the dispensed population of cells and distribute the population of cells into a plurality of perforations; 
 a perforated member comprising the plurality of perforations disposed under and adjacent to the permeate member; 
 a filter disposed under and adjacent to the perforated member and above and adjacent to the permeate member; and 
   a processor configured to operate the automated multi-module cell editing instrument based on user input of a pre-programmed script.   
     
     
         2 . The automated multi-module cell editing instrument of  claim 1 , wherein the automated multi-module cell editing instrument further comprises one or more reagent cartridges. 
     
     
         3 . The automated multi-module cell editing instrument of  claim 2 , wherein the processor controls the dispensing of reagents from one of the reagent cartridges, vectors from one of the reagent cartridges, and editing oligonucleotides from one of the reagent cartridges into the retentate member. 
     
     
         4 . The automated multi-module cell editing instrument of  claim 2 , wherein the processor controls the dispensing of the population of cells into the retentate member. 
     
     
         5 . The automated multi-module cell editing instrument of  claim 1 , wherein the permeate member further comprises one or more permeate member ports configured to supply fluid to and remove fluid from the permeate member. 
     
     
         6 . The automated multi-module cell editing instrument of  claim 5 , wherein the permeate member ports are fluidically-connected to the retentate member, the perforated member, and filter. 
     
     
         7 . The automated multi-module cell editing instrument of  claim 1 , the permeate member, the retentate member, the perforated member, and the filter are coupled together by solvent bonding. 
     
     
         8 . The automated multi-module cell editing instrument of  claim 1 , wherein the retentate member comprises at least one retentate distribution channel(s). 
     
     
         9 . The automated multi-module cell editing instrument of  claim 1 , wherein the retentate member comprises at least one permeate member ridge(s). 
     
     
         10 . The automated multi-module cell editing instrument of  claim 1 , wherein the retentate member comprises at least one permeate member flow director(s). 
     
     
         11 . The automated multi-module cell editing instrument of  claim 1 , wherein the perforated member comprises at least 100,000 perforations, or at least 200,000 perforations, or at least 400,000 perforations. 
     
     
         12 . The automated multi-module cell editing instrument of  claim 1 , wherein the permeate member further comprises ultrasonic tabs. 
     
     
         13 . The automated multi-module cell editing instrument of  claim 1 , further comprising a gasket surrounding the filter. 
     
     
         14 . The automated multi-module cell editing instrument of  claim 1 , wherein the automated multi-module cell editing instrument further comprises a transformation module. 
     
     
         15 . The automated multi-module cell editing instrument of  claim 14 , wherein the transformation module has a flow-through electroporation device for introducing nucleic acids into the population of cells. 
     
     
         16 . The automated multi-module cell editing instrument of  claim 1 , wherein the population of cells is diluted into groups of 2 to 50 cells in the perforated member via substantial Poisson distribution. 
     
     
         17 . The automated multi-module cell editing instrument of  claim 1 , wherein the population of cells is diluted into groups of 2 to 10 cells in the perforated member via substantial Poisson distribution. 
     
     
         18 . The automated multi-module cell editing instrument of  claim 1 , wherein the population of cells is a population of bacterial cells. 
     
     
         19 . The automated multi-module cell editing instrument of  claim 1 , wherein the population of cells is a population of yeast cells. 
     
     
         20 . The automated multi-module cell editing instrument of  claim 1 , wherein the population of cells is a population of mammalian cells.