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

Claim 11:
12. 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;
(c) a λRed recombineering system; and
(d) a first selection marker;

(ii) an editing vector comprising:
(a) a second inducible promoter or a constitutive promoter driving expression of a coding sequence for a recA protein;
(b) the second inducible promoter or the constitutive promoter driving transcription of at least two editing cassettes wherein each editing cassette comprises a gRNA sequence and a donor DNA sequence to be transcribed;
(c) a bacterial origin or replication; and
(d) 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.