CRISPR editing to embed nucleic acid landing pads into genomes of live cells

1. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:

designing and synthesizing a library of editing cassettes comprising landing pads, wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;

inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;

transforming the population of cells with the library of editing vectors;

allowing editing to take place in the population of cells to produce edited cells;

transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for a recombinase or a meganuclease under control of an inducible promoter;

inducing expression of the recombinase or meganuclease to insert the large DNA payloads into the landing pads; and

screening for cells comprising the desired phenotype or genotype.

2. The method of claim 1 , wherein the vectors carrying large DNA payloads comprise a coding sequence for a recombinase, the landing pads comprise a recognition sequence for the recombinase and the large DNA payloads comprise recognition sequences for the recombinase flanking the large DNA payload.

3. The method of claim 2 , wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.

4. The method of claim 2 , wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).

5. The method of claim 1 , wherein the vectors carrying large DNA payloads comprise a coding sequence for a meganuclease, the landing pads comprise a recognition sequence for the meganuclease, and the large DNA payloads further comprise homologous recombination sequences flanking the DNA playloads.

6. The method of claim 5 , wherein the meganuclease belongs to the LAGLIDADG family of nucleases.

7. The method of claim 6 , wherein the meganuclease is I-SceI.

8. The method of claim 6 , wherein the meganuclease is I-CreI.

9. The method of claim 6 , wherein the meganuclease is I-DmoI.

10. The method of claim 1 , wherein the editing cassettes further comprise a barcode.

11. The method of claim 1 , wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette.

12. The method of claim 1 , wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.

13. The method of claim 12 , wherein the selectable marker in the vectors carrying the large DNA payloads is different from a selectable marker in the editing cassettes.

14. The method of claim 1 , wherein the vectors carrying the large DNA payloads comprise the coding sequence of the recombinase or meganuclease under the control of an inducible promoter.

15. The method of claim 14 , wherein the inducible promoter is a pL promoter.

16. The method of claim 14 , wherein the inducible promoter is a pBAD promoter.

17. The method of claim 1 , wherein the vectors carrying large DNA payloads further comprise an origin of replication and a selectable marker.

18. The method of claim 1 , wherein the large DNA payloads are from 100 bp to 100 Kb in length.

19. The method of claim 18 , wherein the large DNA payloads are from 500 bp to 50 Kb in length.

20. The method of claim 1 , wherein the screening step comprises polymerase chain reaction (PCR) analysis with appropriate primer sets; a metabolic test; measurement of transcript level; a phenotypic assay; detection of a protein product using an antibody specific to the protein product;

or DNA sequencing of the integrated large DNA payload.

21. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:

designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a recombinase and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;

inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;

transforming the population of cells with the library of editing vectors;

allowing editing to take place in the population of cells to produce edited cells;

transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the recombinase under control of an inducible promoter;

inducing expression of the recombinase to insert the large DNA payloads into the landing pads; and

screening for cells comprising the desired phenotype or genotype.

22. The method of claim 21 , wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.

23. The method of claim 21 , wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).

24. The method of claim 21 , wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.

25. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:

designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a meganuclease and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;

inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;

transforming the population of cells with the library of editing vectors;

allowing editing to take place in the population of cells to produce edited cells;

transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the meganuclease under control of an inducible promoter;

inducing expression of the meganuclease to insert the large DNA payloads into the landing pads; and

screening for cells comprising the desired phenotype or genotype.

26. The method of claim 25 , wherein the meganuclease belongs to the LAGLIDADG family of nucleases.

27. The method of claim 26 , wherein the meganuclease is I-SceI.

28. The method of claim 26 , wherein the meganuclease is I-CreI.

29. The method of claim 26 , wherein the meganuclease is I-DmoI.

30. The method of claim 25 , wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.