TARGET DNA INTERFERENCE WITH crRNA

• US 20100076057A1
• Filed: 09/23/2009
• Published: 03/25/2010
• Est. Priority Date: 09/23/2008
• Status: Abandoned Application

• Current Assignee: Northwestern University
• Original Assignee: Northwestern University
• Inventors: Sontheimer, Erik J., Marraffini, Luciano A.
• Application Number:
  US12/565,589
• Publication Number:
  US 20100076057A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Abstract

The present invention provides methods, systems, and compositions for interfering with the function and/or presence of a target DNA sequence in a eukaryotic cell (e.g., located in vitro or in a subject) using crRNA and CRISPR-associated (cas) proteins or cas encoding nucleic acids. The present invention also relates to a method for interfering with horizontal gene transfer based on the use of clustered, regularly interspaced short palindromic repeat (CRISPR) sequences.

Claims

1. A method of inhibiting the function and/or presence of a target DNA sequence in a eukaryotic cell comprising:

administering crRNA and one or more cas proteins, or nucleic acid sequences encoding said one or more cas proteins, to a eukaryotic cell comprising a target DNA sequence, wherein said crRNA hybridizes with said target DNA sequence thereby interfering with the function and/or presence of said target DNA sequence.

2. The method of claim 1, wherein said one or more cas proteins comprises Cas3.

3. The method of claim 1, wherein said one or more cas proteins comprise Cas3 and Cse1-5 proteins.

4. The method of claim 1, wherein said interfering with the function and/or presence of said target DNA sequence silences expression of said target DNA sequence.

5. The method of claim 1, wherein said cell is located in a subject.

6. The method of claim 5, wherein said target DNA sequence is a detrimental allele that causes said subject to have a disease or condition.

7. The method of claim 5, wherein said target DNA sequence is located within the genome of said cell.

8. The method of claim 7, wherein said target DNA sequence is located within close proximity to a CRISPR motif sequence.

9. The method of claim 1, further comprising studying the effect on said of interfering with the function and/or presence of said target DNA sequence compared to a control cell.

10. A method of treating or preventing an infection comprising:

administering crRNA and one or more cas proteins, or nucleic acid sequences encoding said one or more cas proteins, to a subject infected by a pathogen or at risk of infection by said pathogen, wherein said crRNA hybridizes to a target DNA sequence from said pathogen thereby interfering with the function and/or presence of said target DNA sequence.

11. The method of claim 10, wherein said interfering with the function and/or presence of said target DNA sequence is fatal to said pathogen.

12. The method of claim 10, wherein said pathogen is a bacterium.

13. The method of claim 10, wherein said pathogen is a virus.

14. The method of claim 10, wherein said pathogen is a fungus.

15. A method of regulating gene transfer within a cell, tissue, or subject comprising inhibiting horizontal gene transfer, wherein clustered, regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (cas) protein-coding genes are configured within the DNA of said cell, tissue, or subject to inhibit horizontal gene transfer into said DNA of said cell, tissue, or subject.

16. The method of claim 15, wherein said subject is an archeabacterium or eubacterium.

17. The method of claim 15, wherein said horizontal gene transfer comprises plasmid conjugation.

18. The method of claim 15, wherein said horizontal gene transfer comprises phage trandsduction.

19. The method of claim 15, wherein said horizontal gene transfer comprises DNA transformation.

20. The method of claim 15, wherein said CRISPR loci comprise 20-50 nucleotide direct repeats of DNA sequence separated by 24-48 nucleotide unique spacers.