Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells

Abstract

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.

1 .- 30 . (canceled) 
     
     
         31 . A method of nucleic acid-guided nuclease editing of five hundred or more exogenous polynucleotides from source cells within heterologous editing cells in a single multiplexed automated operation, comprising:
 (a) providing to a transformation module heterologous editing cells, wherein the heterologous editing cells comprise five hundred or more different target polynucleotides from the source cells in shuttle vector backbones;   (b) providing one or more editing vectors to the heterologous editing cells, wherein the editing vectors comprise a coding sequence for a nuclease, a guide nucleic acid and a DNA donor sequence to the transformation module;   (c) allowing editing to take place in an editing module under conditions that allow the editing vectors to edit the one or more target polynucleotides in the shuttle vectors thereby forming edited shuttle vectors; and   (d) isolating the edited shuttle vectors from the heterologous editing cells;   wherein the transformation module and editing module are all part of a stand-alone automated multi-module cell processing instrument.   
     
     
         32 . The method of  claim 31 , wherein one or more of the five hundred or more different target polynucleotides are selected from the group consisting of single open reading frame coding sequences, a gene sequence from 5′UTR to 3′UTR, and a genetic locus comprising at least 100 kb. 
     
     
         33 . The method of  claim 31 , wherein the source cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, mammalian cells and human cells. 
     
     
         34 . The method of  claim 31 , wherein the shuttle vector backbones are selected from the group consisting of bacterial plasmids, yeast centromeric plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and mammalian artificial chromosomes. 
     
     
         35 . The method of  claim 31 , wherein the heterologous editing cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, mammalian cells and human cells. 
     
     
         36 . The method of  claim 31 , wherein the editing vectors confer edits selected from the group consisting of saturation mutagenesis edits, knockout edits, loss of function edits, gain of function edits, coding sequence edits and noncoding sequence edits. 
     
     
         37 . The method of  claim 31 , wherein one or more of the five hundred or more different target polynucleotides are genomic loci comprising between 1000 nucleotides and 10,000 nucleotides. 
     
     
         38 . The method of  claim 31 , wherein one or more of the five hundred or more different target polynucleotides are genetic loci comprising between 10,000 nucleotides and 100,000 nucleotides. 
     
     
         39 . The method of  claim 31 , wherein the transformation module is a flow-through electroporation (FTEP) device. 
     
     
         40 . The method of  claim 31 , wherein the stand-alone automated multi-module cell processing instrument comprises a growth module. 
     
     
         41 . The method of  claim 40 , wherein the grown module comprises a rotating growth module. 
     
     
         42 . The method of  claim 31 , wherein the stand-alone automated multi-module cell processing instrument further comprises a recovery module. 
     
     
         43 . The method of  claim 31 , wherein the stand-alone automated multi-module cell processing instrument is controlled by a processor. 
     
     
         44 . The method of  claim 31 , wherein the stand-alone automated multi-module cell processing instrument further comprises a reagent cartridge. 
     
     
         45 . The method of  claim 31 , wherein the heterologous editing cells are provided in a growth module prior to step (a). 
     
     
         46 . The method of  claim 45 , wherein a liquid handling system moves the heterologous editing cells from the growth module to the transformation module. 
     
     
         47 . The method of  claim 31 , wherein a liquid handling system moves the heterologous editing cells from the transformation module to the editing module. 
     
     
         48 . The method of  claim 31 , wherein the editing comprises:
 (a) a deletion at least one nucleotide of the five hundred or more different target polynucleotides;   (b) an insertion of at least one nucleotide within the five hundred or more different polynucleotides;   (c) a substitution of at least one nucleotide within the five hundred or more different polynucleotides; or   (d) any combination of (a), (b), and (c).   
     
     
         49 . The method of  claim 31 , wherein the heterologous editing cells in step (d) are living heterologous editing cells. 
     
     
         50 . The method of  claim 33 , wherein the bacterial cells are  Escherichia coli  cells.