GENETIC DEVICE FOR THE CONTROLLED DESTRUCTION OF DNA

• US 20150315576A1
• Filed: 11/01/2013
• Published: 11/05/2015
• Est. Priority Date: 11/01/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Voigt, Christopher, Caliando, Brian
• Application Number:
  US14/440,166
• Publication Number:
  US 20150315576A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid

Abstract

The invention relates to DNA destruction devices and related methods reagents and kits. The DNA destruction devices are useful for achieving target specific DNA destruction in vivo using a system that involves an actuator element and a CRISPR array, under specific regulatory control.

Claims

1. A synthetic DNA destruction device (DDD) comprising a nucleic acid sequence having an actuator sequence under the control of a first regulatory element and a nucleic acid sequence having a Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) array under the control of a second regulatory element.

2. The synthetic DDD of claim 1, wherein the nucleic acid sequence having an actuator sequence and the nucleic acid sequence having a CRISPR array are linked.

3. The synthetic DDD of claim 1, wherein the actuator sequence encodes a DNA targeting/degradation protein.

4. The synthetic DDD of claim 1, wherein the actuator sequence encodes 2-10 DNA targeting/degradation proteins.

5. The synthetic DDD of claim 1, wherein the actuator sequence comprises a CRISPR-associated (cas) gene.

6. The synthetic DDD of claim 1, wherein the cas gene is selected from the group consisting of cas3 and casABCDE.

7. The synthetic DDD of claim 1, wherein the cas gene is six cas genes:

cas3 and casABCDE.

8. The synthetic DDD of claim 5, wherein the cas gene is a single cas gene.

9. The synthetic DDD of claim 1, wherein the CRISPR array includes interspersed sets of target specific spacer sequences between palindromic repeat sequences.

10. The synthetic DDD of claim 9, wherein the interspersed sets of target specific spacer sequences are 29-33 base pairs in length.

20. The synthetic DDD of claim 9, wherein the target specific spacer sequences have an adjacent discriminator sequence.

23. The synthetic DDD of claim 20, wherein the adjacent discriminator sequence is a PAM sequence.

29. The synthetic DDD of claim 9, wherein the target sequence is part of any one or more of a DNA based transposon, bacteriophage nucleic acid, plasmid, and/or chromosome.

30. The synthetic DDD of claim 1, wherein the first regulatory element is a first inducible promoter.

31. The synthetic DDD of claim 1, wherein the second regulatory element is a second inducible promoter.

32. The synthetic DDD of claim 1, wherein the first regulatory element is an activation element that induces expression of the actuator sequence in response to one or more activation signals.

33. The synthetic DDD of claim 1, wherein the second regulatory element is a second activation element that induces the production of a DNA interference RNA from the CRISPR array in the presence of an activation signal.

34. The synthetic DDD of claim 32, wherein the activation signal is a chemical signal.

35. The synthetic DDD of claim 34, wherein the chemical signal is arabinose.

36. The synthetic DDD of claim 32, wherein the activation signal is an environmental signal.

37. The synthetic DDD of claim 1, wherein the first regulatory element is an inhibitory element that maintains the actuator in an inactive state by the presence of an inhibitory signal.

38. The synthetic DDD of claim 37, wherein the inhibitory signal is a chemical signal.

39. The synthetic DDD of claim 37, wherein the inhibitory signal is an environmental signal.

40. The synthetic DDD of claim 1, wherein the first regulatory element is an inhibitory element and an activation element.

41. The synthetic DDD of claim 1, wherein the second regulatory element is an inhibitory element and an activation element.

42. The synthetic DDD of claim 1, wherein the second regulatory element is an inhibitory element that maintains the CRISPR array in an inactive state by the presence of an inhibitory signal.

11-19. -19. (canceled)

21-22. -22. (canceled)

24-28. -28. (canceled)

43-60. -60. (canceled)

61. A method for destroying target specific DNA in a living host cell, comprising,

contacting a living modified host cell having an exogenous nucleic acid actuator sequence under the control of a first regulatory element and an exogenous target specific nucleic acid Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) array under the control of a second regulatory element with a first regulatory signal, wherein the first regulatory signal induces the expression of the actuator sequence to produce an actuator protein, wherein the cell is exposed to a second regulatory signal and the second regulatory signal induces the production of a target specific DNA interference RNA, and wherein the actuator protein and the target specific DNA interference RNA destroy a target DNA in the host cell.

62-80. -80. (canceled)

81. A plasmid comprising a nucleic acid sequence having a Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) array under the control of a regulatory element, wherein the nucleic acid sequence having the CRISPR array has at least two palindromic repeat sequences with a spacer region positioned between the at least two palindromic repeat sequences, wherein the spacer region includes at least two restriction enzyme sequences and optionally a nucleic acid sequence encoding a selectable marker.

82-89. -89. (canceled)