METHODS AND COMPOSITIONS FOR GENERATING CONDITIONAL KNOCK-OUT ALLELES

• US 20150128300A1
• Filed: 06/12/2013
• Published: 05/07/2015
• Est. Priority Date: 06/12/2012
• Status: Abandoned Application

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Warming, Soren, Anderson, Keith R.
• Application Number:
  US14/407,869
• Publication Number:
  US 20150128300A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

The disclosure provides methods and compositions for generating conditional knock-out alleles using donor constructs together with sequence-specific nucleases to generate conditional knock-out alleles. Specifically, the donor construct comprises a 5′ homology region, a 5′ recombinase recognition site, a donor sequence, a 3′ recombinase recognition site, and a 3′ homology region. Further disclosed are the donor sequences each comprises a target sequence having at least one neutral mutation. Different sequence-specific nucleases can be used with the donor constructs are further disclosed.

Claims

1. A method of generating a conditional knock-out allele in a cell comprising a target gene, the method comprising the steps of:

a. introducing into the cell a donor construct, wherein the donor construct comprises a 5′

homology region, a 5′

recombinase recognition site, a donor sequence, a 3′

recombinase recognition site, and a 3′

homology region, wherein the donor sequence comprises a target sequence having at least one neutral mutation; and

b. introducing into the cell a sequence-specific nuclease that cleaves a sequence within the target gene, thereby producing a conditional knock-out allele in the cell.

2. The method of claim 1, wherein the sequence-specific nuclease is a zinc finger nuclease (ZFN).

3. The method of claim 1, wherein the sequence-specific nuclease is a transcription activator-like effector nuclease (TALEN).

4. The method of claim 1, wherein the sequence-specific nuclease is a ZFN dimer that cleaves the target gene only once.

5. The method of claim 1, wherein the sequence-specific nuclease is an RNA-guided nuclease.

6. The method of claim 5, wherein the RNA-guided nuclease is Cas9.

7. The method of claim 1, wherein the sequence-specific nuclease is introduced as a protein, mRNA, or cDNA.

8. The method of claim 1, wherein the recombinase recognition site is a loxP site, an frt site, or a rox site.

9. The method of claim 1, wherein the donor sequence comprises seven silent mutations.

10. The method of claim 1, wherein sequence homology between the donor sequence and the target sequence is 98% or less.

11. The method of claim 10, wherein sequence homology between the donor sequence and the target sequence is 78%.

12. The method of claim 1, wherein the donor construct comprises the sequence of SEQ ID NO:

30, 31, 44, 45, or 46.

13. The method of claim 1, wherein the 5′

homology region comprises at least 1.1 kb and wherein the 3′

homology region comprises at least 1 kb.

14. The method of claim 1, wherein the target gene is selected from the group consisting of Lrp5, Usp10, Nnmt, and Notch3.

15. The method of claim 1, wherein the cell was isolated from a mammal.

16. The method of claim 15, wherein the mammal is selected from the group consisting of mouse, rat, rabbit, hamster, guinea pig, cat, dog, sheep, horse, cow, monkey, and human.

17. The method of claim 1, wherein the cell is a zygote or a pluripotent stem cell.

18. A method of generating a conditional knock-out animal, the method comprising the steps of:

a. introducing a donor construct into a cell comprising a target gene, wherein the donor construct comprises a 5′

homology region, a 5′

recombinase recognition site, a donor sequence, a 3′

recombinase recognition site, and a 3′

homology region, wherein the donor sequence comprises a target sequence having at least one neutral mutation;

b. introducing a sequence-specific nuclease into the cell, wherein the nuclease cleaves the target gene; and

c. introducing the cell into a carrier animal to produce the conditional knock-out animal from the cell.

19. The method of claim 18, wherein the cell is a zygote or a pluripotent stem cell.

30. A non-human conditional knock-out animal prepared according to the method of claim 18.

20. A method of generating a knock-out animal, the method comprising the steps of:

a. introducing a donor construct into a cell comprising a target gene, wherein the donor construct comprises a 5′

homology region, a 5′

recombinase recognition site, a donor sequence, a 3′

recombinase recognition site, and a 3′

homology region, wherein the donor sequence comprises a target sequence having at least one neutral mutation;

b. introducing a sequence-specific nuclease into the cell, wherein the nuclease cleaves the target gene;

c. introducing the cell into a carrier animal to produce a transgenic animal from the transfected cell; and

d. breeding the conditional knock-out animal with a transgenic animal having a transgene encoding a recombinase protein that catalyzes recombination at the 5′ and

3′

recombinase recognition site.

21. The method of claim 20, wherein the cell is a zygote or a pluripotent stem cell.

22. The method of claim 20, wherein the recombinase recognition site is a loxP site and wherein the recombinase is Cre recombinase.

23. The method of claim 20, wherein the recombinase recognition site is an frt site and wherein the recombinase is FLP recombinase.

24. The method of claim 20, wherein the recombinase recognition site is a rox site and wherein the recombinase is Dre recombinase.

25. The method of claim 20, wherein the transgene encoding the recombinase is under the control of a tissue-specific promoter or an inducible promoter.

26. A composition for generating a conditional knock-out allele of a target gene comprising:

a. a donor construct comprising a 5′

homology region, a 5′

recombinase recognition site, a donor sequence, a 3′

recombinase recognition site, and a 3′

homology region, wherein the donor sequence comprises a target sequence having at least one neutral mutation compared to the sequence of the target gene; and

b. a sequence-specific nuclease that recognizes the target gene.

27. The composition of claim 26, wherein the sequence-specific nuclease is selected from the group consisting of ZFN, TALEN, and RNA-guided nuclease.

28. A donor construct comprising the sequence of SEQ ID NO:

30, 31, 44, 45, or 46.

29. A cell comprising the donor construct of claim 28.