US 11,053,515 B2
Pooled genome editing in microbes
Stephen Blaskowski, Oakland, CA (US); Sara da Luz Areosa Cleto, Emeryville, CA (US); Cameron Coates, Oakland, CA (US); Aaron Miller, Berkeley, CA (US); Sharon Nademanee, Alameda, CA (US); Melissa Netwal, Oakland, CA (US); Kedar Patel, Fremont, CA (US); Shawn Szyjka, Martinez, CA (US); Philip Weyman, Alameda, CA (US); Solomon Henry Stonebloom, Alameda, CA (US); Colin Scott Maxwell, Emeryville, CA (US); and Elizabeth Lauren Meier, Oakland, CA (US)
Assigned to Zymergen Inc., Emeryville, CA (US)
Filed by Zymergen Inc., Emeryville, CA (US)
Filed on Mar. 20, 2020, as Appl. No. 16/825,710.
Application 16/825,710 is a continuation of application No. PCT/US2020/021448, filed on Mar. 6, 2020.
Claims priority of provisional application 62/816,035, filed on Mar. 8, 2019.
Prior Publication US 2020/0283802 A1, Sep. 10, 2020
Int. Cl. C12N 15/65 (2006.01); C12N 1/20 (2006.01); C12N 1/16 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 1/16 (2013.01); C12N 1/20 (2013.01); C12N 15/65 (2013.01)] 26 Claims
 
1. 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 constructs comprising one or more editing plasmids, wherein each editing plasmid in the first pool of editing constructs comprises a selection marker gene and one or both of a guide RNA (gRNA) and a repair fragment, wherein the microbial host cells comprise an RNA-guided DNA endonuclease or an RNA-guided DNA endonuclease is introduced into the microbial host cells along with the first pool of editing constructs, and wherein the first pool of editing constructs comprise:
(i) gRNAs that target a same target locus, and at least two different repair fragments, wherein each repair fragment comprises a sequence for one or more genetic edits in or adjacent to the target locus, and wherein sequence for each of the one or more genetic edits lies between homology arms, wherein the homology arms comprise sequence homologous to sequence that flanks the target locus in the genome of the microbial host;
(ii) gRNAs that target at least two different target loci, and at least two different repair fragments, wherein each repair fragment comprises a sequence for one or more genetic edits in or adjacent to the target loci, and wherein sequence for each of the genetic edits lies between homology arms, wherein the homology arms comprise sequence homologous to sequence that flanks the target loci in the genome of the microbial host, wherein the one or more genetic edits are identical; or
(iii) gRNAs that target at least two different target loci, and at least two different repair fragments, wherein each repair fragment comprises a sequence for one or more genetic edits in or adjacent to the target loci, and wherein sequence for each of the genetic edits lies between homology arms, wherein the homology arms comprise sequence homologous to sequence that flanks the target loci in the genome of the microbial host;
(b) 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 an additional pool of editing constructs comprising one or more editing plasmids, wherein each editing plasmid in the additional pool of editing constructs comprises a different selection marker gene than the selection marker gene from the previous step, and wherein the additional pool of editing constructs comprise gRNAs and repair fragments according to any one of step (a)(i)-(iii); and
(d) growing the microbial host cells from the previous step in media selective for microbial host cells expressing the selection marker gene in the editing plasmid in the additional pool of editing constructs, thereby facilitating clearance of the editing plasmid 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).