Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells

Abstract

This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.

We claim: 
     
         1 . A method of transfecting and performing nucleic acid-guided nuclease editing in mammalian cells in an automated editing instrument comprising the steps of:
 providing an automated closed cell editing instrument comprising a growth module and a microfluidic module;   growing mammalian cells in the growth module;   synthesizing a library of editing cassettes off-instrument, wherein each editing cassette comprises a different gRNA and donor DNA pair;   generating, in the microfluidic module, a first plurality of aqueous droplets in a first immiscible carrier fluid, wherein the first plurality of aqueous droplets comprises dNTPs, primers, polymerase and an editing cassette, and wherein each aqueous droplet of the first plurality of aqueous droplets on average comprises one or no editing cassette;   providing, in the microfluidic module, conditions to allow amplification of the editing cassettes in the first plurality of aqueous droplets;   separating aqueous droplets with amplified editing cassettes from aqueous droplets without amplified editing cassettes;   generating, in the microfluidic module, a second plurality of aqueous droplets in a second immiscible carrier fluid, wherein the second plurality of aqueous droplets comprises transfection reagents and a nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion;   adding, in the microfluidic module, the first plurality of aqueous droplets with the amplified editing cassettes to the second immiscible carrier fluid comprising the second plurality of aqueous droplets;   merging, in the microfluidic module, on average one of the first plurality of aqueous droplets with the amplified editing cassette with on average one of the second plurality of aqueous droplets comprising transfection reagents resulting in aqueous droplet reagent bundles;   generating, in the microfluidic module, a third plurality of aqueous droplets in a third immiscible carrier fluid, wherein the third plurality of aqueous droplets comprises the mammalian cells grown in the growth module;   adding, in the microfluidic module, the aqueous droplet reagent bundles to the third immiscible carrier fluid comprising the third plurality of aqueous droplets comprising the mammalian cells;   merging, in the microfluidic module, on average one aqueous droplet reagent bundle with on average one aqueous droplet comprising the mammalian cells to produce merged droplets;   providing, in the microfluidic module, conditions for cell transfection and editing;   transferring the merged droplets to the growth module; and   demulsifying the merged droplets in the growth module resulting in pooled merged droplets.   
     
     
         2 . The method of  claim 1 , wherein the growth module is a rotating growth module, a tangential flow filtration module or a bioreactor module. 
     
     
         3 . The method of  claim 2 , wherein the growth module is a rotating growth module. 
     
     
         4 . The method of  claim 2 , wherein the growth module is a tangential flow filtration module. 
     
     
         5 . The method of  claim 2 , wherein the growth module is a bioreactor. 
     
     
         6 . The method of  claim 1 , wherein the first, second and third immiscible carrier fluids are the same immiscible carrier fluid. 
     
     
         7 . The method of  claim 6 , wherein the first, second and third immiscible carrier fluids are fluorinated oils. 
     
     
         8 . The method of  claim 1 , wherein at least two of the first, second and third immiscible carrier fluids are different immiscible carrier fluids. 
     
     
         9 . The method of  claim 1 , wherein the mammalian cells are grown on microcarriers. 
     
     
         10 . The method of  claim 1 , wherein the merging step is accomplished by a localized electric field, providing locally a chemical that disrupts or destabilizes the surfactant in the second and/or third immiscible carrier fluids, or use of a textured surface in a flow path of a microfluidic channel through which the second and/or third immiscible carrier fluids flow. 
     
     
         11 . The method of  claim 1 , wherein the step of separating aqueous droplets with amplified editing cassettes from aqueous droplets without amplified editing cassettes is accomplished by electrophoresis; dielectricphoresis; acoutstrophoresis; optical sorting; or magnetophoresis. 
     
     
         12 . The method of  claim 11 , wherein the step of separating aqueous droplets with amplified editing cassettes from aqueous droplets without amplified editing cassettes is accomplished by optical sorting. 
     
     
         13 . A method of transfecting and performing nucleic acid-guided nuclease editing in mammalian cells in an automated editing instrument comprising the steps of:
 providing an automated closed cell editing instrument comprising a growth module, a microfluidic module and a solid wall module;   growing mammalian cells in the growth module;   synthesizing a library of editing cassettes off-instrument, wherein each editing cassette comprises a different gRNA and donor DNA pair;   generating, in the microfluidic module, a first plurality of aqueous droplets in a first immiscible carrier fluid, wherein the first plurality of aqueous droplets comprise dNTPs, primers, polymerase, a nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion and an editing cassette, wherein each aqueous droplet of the first plurality of aqueous droplets on average comprises one or no editing cassette;   polymerizing, in the microfluidic module, the first plurality of aqueous droplets resulting in reagent bundle gel beads;   providing, in the microfluidic module, conditions to allow amplification of the editing cassettes in the reagent bundle gel beads;   separating reagent bundle gel beads with amplified editing cassettes from reagent bundle gel beads without amplified editing cassettes;   delivering the reagent bundle gel beads comprising amplified editing cassettes to a solid wall module comprising wells, wherein the wells comprise the mammalian cells and are sized so as to be able to accommodate only one reagent bundle gel bead;   dissolving, in the solid wall module, the reagent bundle gel beads comprising amplified editing cassettes in the wells;   providing transfection reagents to the wells in the solid wall module;   providing conditions to allow transfection and editing in the mammalian cells in the solid wall module;   growing the mammalian cells in the solid wall module;   dislodging the mammalian cells from the wells in the solid wall module; and   pooling the cells.   
     
     
         14 . The method of  claim 13 , wherein the growth module is a rotating growth module, a tangential flow filtration module or a bioreactor module. 
     
     
         15 . The method of  claim 14 , wherein the growth module is a rotating growth module. 
     
     
         16 . The method of  claim 14 , wherein the growth module is a tangential flow filtration module. 
     
     
         17 . The method of  claim 14 , wherein the growth module is a bioreactor. 
     
     
         18 . The method of  claim 13 , wherein the first, second and third immiscible carrier fluids are the same immiscible carrier fluid. 
     
     
         19 . The method of  claim 18 , wherein the first, second and third immiscible carrier fluids are fluorinated oils. 
     
     
         20 . The method of  claim 13 , wherein at least two of the first, second and third immiscible carrier fluids are different immiscible carrier fluids. 
     
     
         21 . The method of  claim 13 , wherein the mammalian cells are grown on microcarriers. 
     
     
         22 . The method of  claim 13 , wherein the step of separating aqueous droplets with amplified editing cassettes from aqueous droplets without amplified editing cassettes is accomplished by electrophoresis; dielectricphoresis; acoutstrophoresis; optical sorting; or magnetophoresis. 
     
     
         23 . The method of  claim 22 , wherein the step of separating aqueous droplets with amplified editing cassettes from aqueous droplets without amplified editing cassettes is accomplished by optical sorting. 
     
     
         24 . The method of  claim 13 , wherein the solid wall module is a solid wall isolation, incubation, and normalization (SWIIN) module. 
     
     
         25 . The method of  claim 24 , wherein the SWIIN module comprises microwells with a volume of approximately 2.5 nl. 
     
     
         26 . The method of  claim 24 , wherein the SWIIN module comprises 200,000 microwells. 
     
     
         27 . The method of  claim 24 , wherein the SWIIN module comprises a heater and a heated cover. 
     
     
         28 . The method of  claim 13 , wherein the reagent bundle gel beads comprise polyacrylamide with disulfide crosslinkers. 
     
     
         29 . The method of  claim 28 , wherein the reagent bundle gel beads are dissolved by exposure of the reagent bundle gel beads to a reducing agent. 
     
     
         30 . The method of  claim 29 , wherein the reducing agent is β-mercaptoethanol, dithiothreitol (DTT), (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), or tris(2-carboxyethyl) phosphine (TCEP).