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 electrocompetent bacteria cells;
- b) providing:
- i) an engine vector comprising:
- a first inducible promoter driving expression of a coding sequence for a CRISPR nuclease;
- a bacterial origin of replication;
- and
- 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;
- the second inducible promoter or the constitutive promoter driving transcription of at least two editing cassettes where each editing cassette comprises a gRNA sequence and a donor DNA sequence to be transcribed;
- a bacterial origin of replication;
- and
- a second selection marker;
- c) transforming the electrocompetent bacterial cells with the engine and editing vectors;
- d) allowing transcription of the recA protein and the at least two editing cassettes from the second inducible promoter or the constitutive promoter;
- e) following transcription of the recA protein and the at least two editing cassettes, inducing transcription of the CRISPR nuclease;
- f) allowing the transformed cells to edit;
- and
- g) pooling the edited cells or selecting small colonies of edited cells.
A method for increasing observed editing in a multiplexed CRISPR nuclease editing system in bacteria comprising:
- a) providing electrocompetent bacteria cells;
- b) providing:
- c) an engine vector comprising:
- i. a first inducible promoter driving expression of a coding sequence for a CRISPR nuclease;
- ii. a bacterial origin of replication;
- iii. a λRed recombineering system;
- and
- iv. a first selection marker;
- d) an editing vector comprising:
- i. a second inducible promoter or a constitutive promoter driving expression of a coding sequence for a recA protein;
- ii. the second inducible promoter or the constitutive promoter driving transcription of at least two editing cassettes where each editing cassette comprises a gRNA sequence and a donor DNA sequence to be transcribed;
- iii. a bacterial origin of replication;
- and
- iv. a second selection marker;
- e) transforming the electrocompetent bacterial cells with the engine and editing vectors;
- f) allowing transcription of the recA protein and the at least two editing cassettes from the second inducible promoter or the constitutive promoter;
- g) following transcription of the recA protein and the at least two editing cassettes, inducing transcription of the CRISPR nuclease;
- h) allowing the transformed cells to edit;
- i) growing the edited cells in colonies until the cells enter the stationary phase of growth and cell colonies become normalized;
- and
- j) pooling the normalized cells.
Dependencies are inferred from the claim text.