Methods for increasing observed editing in bacteria
AssigneeInscripta, Inc.
InventorsTian Tian, Eileen Spindler, Charles Johnson, Clint Davis
Life of this patent
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Abstract
The present disclosure relates to methods for increasing observed editing rates in the surviving bacteria cells. The compositions and methods presented herein in combination lead to a phenomenon of “edit or die.” Although less cells survive plating and editing, a large percentage of cells that do survive are multiple editors. In one experiment it was found that if a cell survives transformation, plating, and editing, 75% of the surviving cells are multiple editors; that is, 75% of the surviving cells were simultaneously edited with edits at two or more different locations within the bacterial genome.
Claims20
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.
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.
Dependencies are inferred from the claim text.