Methods and compositions for using zinc finger endonucleases to enhance homologous recombination

• US 20030232410A1
• Filed: 03/20/2003
• Published: 12/18/2003
• Est. Priority Date: 03/21/2002
• Status: Abandoned Application

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Aspland, Simon Eric, Liljedahl, Monika, Segal, David J.
• Application Number:
  US10/395,816
• Publication Number:
  US 20030232410A1
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Embodiments relate to methods of generating a genetically modified cell. The methods can include providing a primary cell containing an endogenous chromosomal target DNA sequence in which it is desired to have homologous recombination occur. The methods also can include providing a zinc finger endonuclease (ZFE) that includes an endonuclease domain that cuts DNA, and a zinc finger domain that includes a plurality of zinc fingers that bind to a specific nucleotide sequence within the endogenous chromosomal target DNA in the primary cell. Further, the methods can include contacting the endogenous chromosomal target DNA sequence with the zinc finger endonuclease in the primary cell such that the zinc finger endonuclease cuts both strands of a nucleotide sequence within the endogenous chromosomal target DNA sequence in the primary cell, thereby enhancing the frequency of homologous recombination in the endogenous chromosomal target DNA sequence. The methods also include providing a nucleic acid comprising a sequence homologous to at least a portion of said endogenous chromosomal target DNA such that homologous recombination occurs between the endogenous chromosomal target DNA sequence and the nucleic acid.

Claims

1. A method of generating a genetically modified cell, comprising:

providing a primary cell containing an endogenous chromosomal target DNA sequence in which it is desired to have homologous recombination occur;

providing a zinc finger endonuclease comprising an endonuclease domain that cuts DNA, and a zinc finger domain comprising a plurality of zinc fingers that bind to a specific nucleotide sequence within said endogenous chromosomal target DNA in said primary cell;

contacting said endogenous chromosomal target DNA sequence with said zinc finger endonuclease in said primary cell such that said zinc finger endonuclease cuts both strands of a nucleotide sequence within said endogenous chromosomal target DNA sequence in said primary cell, thereby enhancing the frequency of homologous recombination in said endogenous chromosomal target DNA sequence; and

providing a nucleic acid comprising a sequence homologous to at least a portion of said endogenous chromosomal target DNA such that homologous recombination occurs between said endogenous chromosomal target DNA sequence and said nucleic acid.

2. The method of claim 1, wherein said zinc finger endonuclease further comprises a protein tag to purify the resultant protein.

3. The method of claim 2, wherein said protein tag is selected from the group consisting of HA tag, FLAG-tag, GST-tag, c-myc, and His-tag.

4. The method of claim 1, wherein said contacting comprises transfecting said primary cell with a vector comprising a cDNA encoding said zinc finger endonuclease and expressing a zinc finger endonuclease protein in said primary cell.

5. The method of claim 1, wherein said contacting comprises injecting a zinc finger endonuclease protein into said primary cell.

6. The method of claim 1, wherein said endonuclease domain is selected from the group consisting of HO endonuclease and Fok I endonuclease.

7. The method of claim 1, wherein said zinc finger domain that binds to a specific nucleotide sequence within said endogenous chromosomal target DNA comprises five or more zinc fingers.

8. The method of claim 1, wherein said zinc finger domain that binds to a specific nucleotide sequence within said endogenous chromosomal target DNA comprises three or more zinc fingers.

9. The method of claim 1, wherein each of said plurality of zinc fingers binds to the sequence G/ANN.

10. The method of claim 1, wherein said cell is from an organism selected from the group consisting of a plant, a mammal, a marsupial, an avian, and teleost fish.

11. The method of claim 10, wherein said mammal is selected from the group consisting of a human, a non-human primate, a sheep, a goat, a cow, a rat and a pig.

12. The method of claim 10, wherein said mammal is a mouse.

13. The method of claim 10, wherein said teleost fish is a zebrafish.

14. The method of claim 10, wherein the avian is a chicken or a turkey.

15. The method of claim 1, wherein said primary cell is from an organism in which totipotent stem cells are not available.

16. A method of designing a sequence specific zinc finger endonuclease capable of cleaving DNA at a specific location, comprising:

identifying a first unique endogenous chromosomal nucleotide sequence adjacent to a second nucleotide sequence at which it is desired to introduce a double-stranded cut; and

designing a combination of sequence specific zinc finger endonucleases that are capable of cleaving DNA at a specific location, said zinc finger endonucleases comprising a plurality of zinc fingers which bind to said unique endogenous chromosomal nucleotide sequence and an endonuclease which generates a double-stranded cut at said second nucleotide sequence.

17. A zinc finger endonuclease for cutting a specific DNA sequence to enhance the rate of homologous recombination, comprising:

an endonuclease domain;

a zinc finger domain specific for an endogenous chromosomal DNA sequence; and

a purification tag.

18. The zinc finger endonuclease of claim 17, wherein said endonuclease domain is selected from the group consisting of HO endonuclease and Fok I endonuclease.

19. The zinc finger endonuclease of claim 17, wherein said zinc finger domain specific for an endogenous chromosomal DNA sequence comprises six zinc fingers.

20. The zinc finger endonuclease of claim 19, wherein said zinc finger domain specific for an endogenous chromosomal DNA sequence comprises at least five zinc fingers.

21. The zinc finger endonuclease of claim 19, wherein said zinc finger domain specific for said endogenous chromosomal DNA sequence comprises three zinc fingers.

22. The zinc finger endonuclease of claim 17, wherein said purification tag is selected from the group consisting of HA tag, FLAG-tag, GST-tag, c-myc, and His-tag.

23. A method of generating a genetically modified animal in which a desired nucleic acid has been introduced, comprising:

obtaining a primary cell comprising an endogenous chromosomal target DNA sequence into which it is desired to introduce said nucleic acid;

generating a double-stranded cut within said endogenous chromosomal target DNA sequence with a zinc finger endonuclease comprising a zinc finger domain that binds to an endogenous target nucleotide sequence within said target sequence and an endonuclease domain;

introducing an exogenous nucleic acid comprising a sequence homologous to at least a portion of said endogenous chromosomal target DNA into said primary cell under conditions which permit homologous recombination to occur between said exogenous nucleic acid and said endogenous chromosomal target DNA; and

generating an animal from said primary cell in which homologous recombination has occurred.

24. The method of claim 23, wherein said zinc finger domain comprises a plurality of zinc fingers.

25. The method of claim 23, wherein said zinc finger domain comprises at least 3 zinc fingers.

26. The method of claim 23, wherein said zinc finger domain comprises at least 5 zinc fingers.

27. The method of claim 23, wherein said animal is selected from the group consisting of a mammal, a marsupial, an avian, and teleost fish.

28. The method of claim 27, wherein said mammal is selected from the group consisting of a human, a non-human primate, a sheep, a goat, a cow, a rat and a pig.

29. The method of claim 27, wherein said mammal is a mouse.

30. The method of claim 27, wherein said teleost fish is a zebrafish.

31. The method of claim 27, wherein the avian is a chicken or a turkey.

32. The method of claim 23, wherein said homologous nucleic acid comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence which disrupts a gene after homologous recombination, a nucleotide sequence which replaces a gene after homologous recombination, a nucleotide sequence which introduces a point mutation into a gene after homologous recombination, and a nucleotide sequence which introduces a regulatory site after homologous recombination.

33. The method of claim 32, wherein said regulatory site comprises a LoxP site.

34. A genetically modified animal made according to the method of claim 23.

35. A method of generating a genetically modified plant in which a desired nucleic acid has been introduced, comprising:

obtaining a plant cell comprising an endogenous target DNA sequence into which it is desired to introduce said nucleic acid;

generating a double-stranded cut within said endogenous target DNA sequence with a zinc finger endonuclease comprising a zinc finger domain that binds to an endogenous target nucleotide sequence within said target sequence and an endonuclease domain;

introducing an exogenous nucleic acid comprising a sequence homologous to at least a portion of said endogenous target DNA into said plant cell under conditions which permit homologous recombination to occur between said exogenous nucleic acid and said endogenous target DNA; and

generating a plant from said plant cell in which homologous recombination has occurred.

36. A genetically modified plant made according to the method of claim 35.