US 11,891,609 B2
Methods for increasing observed editing in bacteria
Tian Tian, Boulder, CO (US); Eileen Spindler, Boulder, CO (US); Charles Johnson, Boulder, CO (US); and Clint Davis, Boulder, CO (US)
Assigned to Inscripta, Inc., Pleasanton, CA (US)
Filed by Inscripta, Inc., Boulder, CO (US)
Filed on Feb. 25, 2022, as Appl. No. 17/680,289.
Application 17/680,289 is a continuation of application No. 17/536,067, filed on Nov. 28, 2021, granted, now 11,319,542.
Application 17/536,067 is a continuation of application No. 16/952,024, filed on Nov. 18, 2020, granted, now 11,203,762, issued on Dec. 21, 2021.
Claims priority of provisional application 62/937,289, filed on Nov. 19, 2019.
Prior Publication US 2022/0177899 A1, Jun. 9, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/74 (2006.01); C12N 15/113 (2010.01); C12N 1/20 (2006.01); C12N 9/22 (2006.01)
CPC C12N 15/74 (2013.01) [C12N 1/20 (2013.01); C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 2310/20 (2017.05)] 20 Claims
 
1. A method for increasing observed editing in a multiplexed CRISPR nuclease editing system in bacteria comprising:
(a) providing bacteria cells, wherein each of the bacteria cells comprises
(i) an engine vector comprising:
(A) a first inducible promoter driving expression of a coding sequence for a CRISPR nuclease;
(B) a bacterial origin of replication; and
(C) a first selection marker; and
(ii) an editing vector comprising a second inducible promoter or a constitutive promoter driving expression of a coding sequence for a recA protein, wherein the second inducible promoter or the constitutive promoter drives transcription of at least two editing cassettes, wherein each editing cassette comprises:
(A) a gRNA sequence and a donor DNA sequence to be transcribed;
(B) a bacterial origin or replication; and
(C) a second selection marker;
(b) allowing transcription of the recA protein and the at least two editing cassettes from the second inducible promoter or the constitutive promoter; and
(c) following transcription of the recA protein and the at least two editing cassettes, inducing transcription of the CRISPR nuclease to produce edited cells.