Telomerator-a tool for chromosome engineering

Patent metadata

Field	Value
Publication number	US-10047366-B2
Application number	US-201414772832-A
Country	US
Kind code	B2
Filing date	Mar 6, 2014
Priority date	Mar 6, 2013
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

Abstract

The present invention relates to the field of chromosome engineering. More specifically, the present invention provides methods and compositions useful for inducibly linearizing circular DNA molecules in vivo in yeast. In one embodiment, a comprises a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding a selectable marker comprises an intron comprising an endonuclease recognition site flanked by telomere seed sequences.

First claim

We claim:

1. A telomerator cassette comprising a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding a selectable marker is interrupted by an intron comprising an endonuclease recognition site flanked by telomere seed sequences.

2. The telomerator cassette of claim 1 , wherein the intron is the ACT1 intron.

3. The telomerator cassette of claim 1 , wherein the selectable marker is an auxotrophic marker.

4. The telomerator cassette of claim 3 , wherein the auxotrophic marker is the URA3 gene.

5. The telomerator cassette of claim 1 , wherein the endonuclease recognition site is specific for I-SceI.

6. The telomerator cassette of claim 5 , wherein the I-SceI endonuclease recognition site comprises SEQ ID NO:2.

7. The telomerator cassette of claim 1 , wherein the telomere seed sequences comprise SEQ ID NO:1 and SEQ ID NO:3.

8. A circular chromosome comprising the telomerator cassette of claim 1 .

9. A eukaryotic host cell comprising the circular chromosome of claim 8 .

10. The eukaryotic host cell of claim 9 , wherein the eukaryote is yeast.

11. The eukaryotic host cell of claim 10 , wherein the yeast is S. cerevisiae.

12. The eukaryotic host cell of claim 9 , wherein the host cell comprises a vector encoding the endonuclease under the control of an inducible promoter.

13. A telomerator cassette comprising a nucleic acid that encodes the auxotrophic marker URA3, wherein the nucleic acid is interrupted by an intron that comprises the I-SceI endonuclease recognition site flanked by telomere seed sequences.

14. The telomerator cassette of claim 13 , wherein the telomere seed sequences comprise SEQ ID NO:1 and SEQ ID NO:3.

15. The telomerator cassette of claim 13 , wherein the I-SceI endonuclease recognition site comprises SEQ ID NO:2.

16. A circular chromosome comprising the telomerator cassette of claim 13 .

17. A yeast host cell comprising the circular chromosome of claim 16 .

18. The yeast host cell of claim 17 , wherein the yeast host cell comprises a vector encoding the I-SceI endonuclease under the control of an inducible promoter.

19. The yeast host cell of claim 18 , wherein the inducible promoter is the GAL1 promoter.

20. A yeast host cell comprising:
a. a circular chromosome comprising a telomerator cassette encoding a selectable marker, wherein the nucleic acid encoding the selectable marker is interrupted by an intron that comprises an endonuclease recognition site flanked by telomere seed sequences; and
b. a vector encoding the endonuclease under the control of an inducible promoter.

21. The yeast host cell of claim 20 , wherein the selectable marker is an auxotrophic marker.

22. The yeast host cell of claim 21 , wherein the auxotrophic marker is the URA3 gene.

23. The yeast host cell of claim 20 , wherein the endonuclease recognition site is specific for I-SceI.

24. The yeast host cell of claim 23 , wherein the I-SceI endonuclease recognition site comprises SEQ ID NO:2.

25. The yeast host cell of claim 20 , wherein the telomere seed sequences comprise SEQ ID NO:1 and SEQ ID NO:3.

26. A method for engineering a circular chromosome capable of being inducibly linearized comprising the step of integrating a telomerator cassette into a circular chromosome, wherein the telomerator cassette comprises a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding a selectable marker is interrupted by an intron comprising an endonuclease recognition site flanked by telomere seed sequences.

27. The method of claim 26 , wherein the integrating step is accomplished by transforming a telomerator cassette into a host cell comprising the circular chromosome.

28. The method of claim 27 , wherein the host cell comprises a vector encoding the endonuclease under the control of an inducible promoter.

29. A telomerator cassette comprising a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding the selectable marker is interrupted by an endonuclease recognition site flanked by telomere seed sequences such that the selectable marker retains its function.

30. The telomerator cassette of claim 29 , wherein the selectable marker is an auxotrophic marker.

31. The telomerator cassette of claim 30 , wherein the auxotrophic marker is the URA3 gene.

32. The telomerator cassette of claim 29 , wherein the endonuclease recognition site is specific for I-SceI.

33. The telomerator cassette of claim 32 , wherein the I-SceI endonuclease recognition site comprises SEQ ID NO:2.

34. The telomerator cassette of claim 29 , wherein the telomere seed sequences comprise SEQ ID NO:1 and SEQ ID NO:3.

35. A circular chromosome comprising the telomerator cassette of claim 29 .

36. A eukaryotic host cell comprising the circular chromosome of claim 35 .

Assignees

• Univ Johns Hopkins

Inventors

• Boeke Jef D
• Mitchell Leslie

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/65

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/905

  in yeast · CPC title