Methods for increasing observed editing in bacteria

1. 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;

a second inducible promoter or a constitutive promoter driving expression of a coding sequence for a recA protein; and

a first selection marker; and

ii) an editing vector comprising:

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.

2. The method of claim 1 , wherein the nuclease is MAD7.

3. The method of claim 1 , wherein the nuclease is Cas9.

4. The method of claim 1 , wherein the coding sequence for the recA protein is a coding sequence for a recA fusion protein.

5. The method of claim 4 , wherein the recA fusion protein is a recA-srpR fusion protein.

6. The method of claim 5 , wherein the recA-srpR fusion protein comprises an in-frame fusion protein comprising a coding sequence of the srpR protein at an N-terminal portion of the in-frame fusion protein and the coding sequence for the recA protein coding sequence at a C-terminal portion of the in-frame fusion protein.

7. The method of claim 1 , wherein the engine vector comprises a coding sequence for c1857 and the inducible promoter is a pL promoter driving expression of the nuclease.

8. The method of claim 1 , wherein the engine vector further comprises coding sequences for a λRed recombineering system.

9. The method of claim 1 , wherein the second inducible promoter driving transcription of the at least two editing cassettes is a pL inducible promoter.

10. The method of claim 1 , wherein the transforming step is performed by electroporation, lipofection, optoporation, microprecipitation, or bead transfection.

11. The method of claim 1 , wherein the first and second selection markers are different selection markers.

12. The method of claim 1 , further comprising the steps of, after the pooling or selecting step:

h) making the edited bacterial cells electrocompetent;

i) providing a second editing vector comprising 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 third selection marker;

j) transforming the electrocompetent bacterial cells with the second editing vector;

k) allowing transcription of the recA protein and the at least two editing cassettes from the second inducible promoter or the constitutive promoter;

l) following transcription of the recA protein and the at least two editing cassettes, inducing transcription of the CRISPR nuclease;

m) allowing the transformed cells to edit; and

n) pooling the twice-edited cells.

13. 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 second inducible promoter or a constitutive promoter driving expression of a coding sequence for a recA protein;

iv. a λRed recombineering system; and

v. a first selection marker;

d) an editing vector comprising:

i. a second inducible promoter or a 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;

ii. a bacterial origin of replication; and

iii. 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.

14. The method of claim 13 , wherein the nuclease is MAD7.

15. The method of claim 13 , wherein the nuclease is Cas9.

16. The method of claim 13 , wherein the coding sequence for the recA protein is a coding sequence for a recA fusion protein.

17. The method of claim 16 , wherein the recA fusion protein is a recA-srpR fusion protein.

18. The method of claim 17 , wherein the recA-srpR fusion protein comprises an in-frame fusion protein comprising a coding sequence of the srpR protein at an N-terminal portion of the in-frame fusion protein and the coding sequence for the recA protein coding sequence at a C-terminal portion of the in-frame fusion protein.

19. The method of claim 13 , wherein the engine vector comprises a coding sequence for c1857 and an inducible pL promoter drives expression of the nuclease and the at least two editing cassettes.

20. The method of claim 13 , wherein the first and second selection markers are different selection markers.