Genome engineering

Patent metadata

Field	Value
Publication number	US-11306328-B2
Application number	US-201414319498-A
Country	US
Kind code	B2
Filing date	Jun 30, 2014
Priority date	Jul 26, 2013
Publication date	Apr 19, 2022
Grant date	Apr 19, 2022

Abstract

Methods are provided for altering target DNA in a cell genetically modified to express a Cas 9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner. Methods include introducing into the cell a first foreign nucleic acid encoding a donor nucleic acid sequence, introducing into the cell from media surrounding the cell the guide RNA complementary to the target DNA and which guides the Cas 9 enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, wherein the donor nucleic acid sequence is expressed, wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell.

First claim

The invention claimed is:

1. A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising
(a) introducing into the cell a DNA donor nucleic acid sequence,
(b) introducing into the cell a guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is in native form or is modified from native form to include a 5′ Cap structure or is modified from native form by treating with phosphatase,
wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and
repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.

2. The method of claim 1 wherein the guide RNA is between about 10 and about 500 nucleotides.

3. The method of claim 1 wherein the guide RNA is between about 20 and about 100 nucleotides.

4. The method of claim 1 wherein the RNA is a crRNA-tracrRNA fusion.

5. The method of claim 1 wherein the donor nucleic acid sequence is inserted by homologous recombination.

6. The method of claim 1 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.

7. The method of claim 1 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNAs to produce multiple alterations to the DNA in the cell.

8. The method of claim 1 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

9. The method of claim 1 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

10. The method of claim 1 wherein the Cas9 enzyme is S. pyogenes Cas9.

11. The method of claim 1 wherein the eukaryotic cell is a human induced pluripotent stem cell.

12. The method of claim 1 wherein the guide RNA is about 100 nucleotides.

13. The method of claim 1 wherein the eukaryotic cell is a stem cell.

14. A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising
(a) introducing into the cell a DNA donor nucleic acid sequence,
(b) introducing into the cell guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is modified from native form to include a 5′ Cap structure,
wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and
repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.

15. The method of claim 14 wherein the guide RNA is between about 10 and about 500 nucleotides.

16. The method of claim 14 wherein the guide RNA is between about 20 and about 100 nucleotides.

17. The method of claim 14 wherein the RNA is a crRNA-tracrRNA fusion.

18. The method of claim 14 wherein the donor nucleic acid sequence is inserted by homologous recombination.

19. The method of claim 14 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.

20. The method of claim 14 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNA to produce multiple alterations to the DNA in the cell.

21. The method of claim 14 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

22. The method of claim 14 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

23. The method of claim 14 wherein the eukaryotic cell is a human induced pluripotent stem cell.

24. The method of claim 14 wherein the guide RNA is about 100 nucleotides.

25. The method of claim 14 wherein the eukaryotic cell is a stem cell.

26. A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising
(a) introducing into the cell a DNA donor nucleic acid sequence,
(b) introducing into the cell guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is modified from native form by treating with phosphatase,
wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and
repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.

27. The method of claim 26 wherein the guide RNA is between about 10 and about 500 nucleotides.

28. The method of claim 26 wherein the guide RNA is between about 20 and about 100 nucleotides.

29. The method of claim 26 wherein the RNA is a crRNA-tracrRNA fusion.

30. The method of claim 26 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNA to produce multiple alterations to the DNA in the cell.

31. The method of claim 26 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

32. The method of claim 26 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

33. The method of claim 26 wherein the eukaryotic cell is a human induced pluripotent stem cell.

34. The method of claim 26 wherein the guide RNA is about 100 nucleotides.

35. The method of claim 26 wherein the eukaryotic cell is a stem cell.

Assignees

• Harvard College

Inventors

• Church George M
• Yang Luhan
• Guell Marc
• Yang Joyce Lichi

Classifications

• C12N2830/003

  tet inducible · CPC title

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title