Patent ID: 11891609
Assignee: INSCRIPTA, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.