Pooled genome editing in microbes

Abstract

The present invention relates to methods for editing the genome of a microbial host cell in one or more rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in a pooled and/or iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Compositions and kits for performing the methods are also disclosed.

1 )- 98 ) (canceled) 
     
     
         99 . A method for generating one or more genetically modified microbial strains, the method comprising:
 (a) introducing into a population of microbial host cells a first pool of editing plasmids, wherein each editing plasmid in the first pool comprises an identical selection marker gene and at least one repair fragment, wherein each repair fragment comprises one or more genetic edits that are each flanked by homology arms, wherein the homology arms comprise sequence complementary to one or more target loci in the genome of the microbial host cells, and wherein the first pool of editing plasmids comprises at least two different repair fragments;   (c) growing the microbial host cells from the previous step in a media selective for microbial host cells expressing the selection marker gene on the editing plasmids from the previous step, thereby producing a population of edited microbial host cells;   (c) introducing into the population of edited microbial host cells from the previous step one or more editing plasmids from an additional pool of editing plasmids, wherein each editing plasmid in the additional pool of editing plasmids comprises an identical selection marker gene that is different than the selection marker gene from the previous step, and wherein the additional pool of editing plasmids comprises at least two different repair fragments, wherein each repair fragment comprises sequence for one or more genetic edits that are each flanked by homology arms that comprise sequence complementary to the one or more target loci in the genome of the microbial host; and   (d) growing the microbial host cells from the previous step in media selective for microbial host cells expressing the selection marker genes in the editing plasmids in the additional pool of editing plasmids, thereby facilitating clearance of the editing plasmids with the different selection marker gene from the population of edited microbial host cells and generating an additional population of edited microbial host cells; wherein a counterselection is not performed before step (c).   
     
     
         100 . The method of  claim 99 , wherein the microbial host cells comprise one or more site-specific restriction enzymes capable of cleaving DNA at the one or more target loci to induce homology dependent repair. 
     
     
         101 . The method of  claim 99 , wherein different editing plasmids are introduced into sub-populations of the microbial host cells of steps (a) or (c). 
     
     
         102 . The method of  claim 99 , wherein the at least two different repair fragments are present on the same editing plasmid. 
     
     
         103 . The method of  claim 99 , wherein the population of edited microbial host cells of steps (b) or (d) comprises individual cells having one genomic edit and individual cells comprising multiple genomic edits. 
     
     
         104 . The method of  claim 99 , further comprising step (e) repeating steps (c) and (d) one or more time for one or more additional rounds of gene editing. 
     
     
         105 . The method of  claim 99 , wherein each repair fragment comprises multiple genetic edits. 
     
     
         106 . The method of  claim 99 , wherein each editing plasmid in the first and the additional pool of editing plasmids comprises the same origin of replication compared to each editing plasmid in the each other or additional pool of editing plasmids previously combined with the microbial host cells. 
     
     
         107 . The method of  claim 99 , wherein each editing plasmid in the first and the additional pool of editing plasmids comprises a same class origin of replication compared to each editing plasmid in the each other or additional pool of editing plasmids previously combined with the microbial host cells. 
     
     
         108 . The method of  claim 100 , wherein each of the one or more site-specific restriction enzymes is selected from the group consisting of an RNA-guided endonuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), and a zinc-finger nuclease (ZFN). 
     
     
         109 . The method of  claim 100 , wherein each of the site-specific restriction enzymes is expressed from a nucleic acid in the microbial host cell or introduced into the cell as protein. 
     
     
         110 . The method of  claim 99 , wherein the one or more genetic edits is selected from the group consisting of an insertion, a deletion, a single nucleotide polymorphism, a genome shuffling, a large scale deletion, a genomic edit, a plasmid edit, and multiple edits, or any combination thereof. 
     
     
         111 . The method of  claim 104 , wherein the at least one repair fragment introduced in the additional pool of editing plasmids is different from the editing plasmids in the previous introducing step. 
     
     
         112 . The method of  claim 99 , wherein the microbial host cell is a eukaryotic cell or prokaryotic cell. 
     
     
         113 . The method of  claim 112 , wherein the eukaryotic host cell is a yeast cell. 
     
     
         114 . The method of  claim 113 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         115 . The method of  claim 113 , wherein the eukaryotic cell is a filamentous fungus. 
     
     
         116 . The method of  claim 115 , wherein the filamentous fungus is  Aspergillus niger.    
     
     
         117 . The method of  claim 112 , wherein the prokaryotic host cell is  Escherichia coli  ( E. coli ). 
     
     
         118 . The method of  claim 112 , wherein the prokaryotic host cell is  Corynebacterium glutamicum  ( C. glutamicum ).