US 11,053,506 B2
Iterative 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. 16/825,683.
Application 16/825,683 is a continuation of application No. PCT/US2020/021472, filed on Mar. 6, 2020.
Claims priority of provisional application 62/816,031, filed on Mar. 8, 2019.
Prior Publication US 2020/0283780 A1, Sep. 10, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/70 (2006.01); C12N 15/113 (2010.01); C12N 9/22 (2006.01); C12N 15/81 (2006.01); C12N 15/77 (2006.01); C12N 15/80 (2006.01)
CPC C12N 15/70 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/77 (2013.01); C12N 15/80 (2013.01); C12N 15/81 (2013.01); C12N 2310/20 (2017.05); C12N 2800/10 (2013.01); C12N 2800/80 (2013.01)] 27 Claims
 
1. A method for iteratively editing a microbial host cell's genome, the method comprising:
(a) introducing into the microbial host cell an editing plasmid, a first guide RNA (gRNA) and a first repair fragment, wherein the gRNA comprises a sequence complementary to a first locus in the genome of the microbial host cell, wherein the first repair fragment comprises homology arms separated by a sequence for a genetic edit in or adjacent to the first locus in the genome of the microbial host cell, wherein the homology arms comprise sequence homologous to sequence that flanks the first locus in the genome of the microbial host cell, wherein the editing plasmid comprises a selection marker gene and at least one or both of a gene encoding the first gRNA and the first repair fragment, and wherein:
(i) the microbial host cell comprises an RNA-guided DNA endonuclease; or
(ii) an RNA-guided DNA endonuclease is introduced into the microbial host cell along with the editing plasmid;
(b) growing the microbial host cell from step (a) in media selective for microbial host cells expressing the selection marker gene in the editing plasmid, thereby creating an edited microbial host cell;
(c) introducing into the edited microbial host cell of the previous step an additional editing plasmid, an additional gRNA and an additional repair fragment, wherein the additional gRNA comprises sequence complementary to an additional locus in the genome of the edited microbial host cell, wherein the additional repair fragment comprises homology arms separated by a sequence for a genetic edit in or adjacent to the additional locus in the genome of the edited microbial host cell, wherein the homology arms comprise sequence homologous to sequence that flanks the additional locus in the genome of the edited microbial host cell, wherein the additional editing plasmid comprises a selection marker gene that is different from the selection marker gene introduced in the previous introducing step, and wherein the additional editing plasmid comprises at least one or both of a gene encoding the additional gRNA and the additional repair fragment, thereby iteratively editing the microbial host cell genome; and
(d) growing the edited microbial host cell from the previous step in media selective for microbial host cells expressing the selection marker gene in the additional editing plasmid of the previous step, thereby clearing the editing plasmids lacking the selection marker gene in the additional editing plasmid of the previous step from the edited microbial host cell from the previous step; wherein a counterselection is not performed before step (c).