Porcine reproductive and respiratory syndrome virus resistant animals
The present invention generally relates to genetically modified swine wherein at least one allele of a SIGLEC1 gene has been inactivated and/or at least one allele of a CD163 gene has been inactivated. Genetically modified swine having both alleles of the SIGLEC1 gene and/or both alleles CD163 gene inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSV). Methods for producing such transgenic swine are also provided.
1. A genetically modified swine comprising an alteration in a CD163 gene sequence, wherein the alteration comprises an alteration in a portion of the CD163 gene sequence that encodes the SRCR5 domain of the CD163 protein, and wherein the swine exhibits increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV).
2. The genetically modified swine of claim 1 , wherein the alteration comprises an insertion, a deletion, a point mutation, a frame-shift mutation, a nonsense mutation, a missense mutation, or a combination of any thereof.
3. The genetically modified swine of claim 2 , wherein the alteration comprises a deletion.
4. The genetically modified swine of claim 3 , wherein the deletion results in deletion of the SRCR5 domain of the CD163 protein.
5. The genetically modified swine of claim 1 , wherein the alteration comprises a replacement of all or a portion of the CD163 gene sequence that encodes the SRCR5 domain with a different nucleic acid sequence.
6. The genetically modified swine of claim 5 , wherein the alteration comprises a replacement of the portion of the CD163 gene sequence that encodes the SRCR5 domain with a nucleic acid sequence that encodes the SRCR8 domain of CD163L.
7. The genetically modified swine of claim 1 , wherein the alteration comprises a deletion of the portion of the CD163 gene sequence that encodes the SRCR5 domain, with no replacement of any of the deleted sequence with a different nucleic acid sequence.
8. The genetically modified swine of claim 2 , wherein the alteration comprises a point mutation that alters the structure of the SRCR5 domain.
9. The genetically modified swine of claim 1 , wherein the alteration results in introduction of a stop codon in the portion of the CD163 gene sequence that encodes the SRCR5 domain of the CD163 protein.
10. The genetically modified swine of claim 1 , wherein the alteration results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).
11. The genetically modified swine of claim 1 , wherein the swine does not comprise a mutation in either allele of a SIGLEC1 gene.
12. Progeny of the genetically modified swine of claim 1 , wherein the progeny comprise the alteration in the CD163 gene sequence.
13. A method for producing a genetically modified swine of claim 1 , the method comprising:
introducing a site-specific nuclease into a porcine cell, the nuclease being adapted to bind to a target sequence in an allele of the CD163 gene; and
incubating the cell under conditions that permit the nuclease to act upon DNA at or near the target sequence and thereby induce recombination, homology-directed repair, or nonhomologous end joining at or near the target sequence.
14. The method of claim 13 , wherein the site-specific nuclease comprises a zinc-finger nuclease or a TAL effector nuclease.
15. The method of claim 13 , wherein the alteration comprises a deletion of the portion of the CD163 gene sequence that encodes the SRCR5 domain, with no replacement of any of the deleted sequence with a different nucleic acid sequence.
16. The method of claim 13 , wherein the alteration results in introduction of a stop codon in the portion of the CD163 gene sequence that encodes the SRCR5 domain of the CD163 protein.
17. The method of claim 13 , wherein the method further comprises generating an animal from the cell.
18. The method of claim 17 , wherein the method further comprises mating a female animal produced by the method of claim 17 with a male animal produced by the method of claim 17 to produce F1 progeny; and
screening the F1 progeny to identify animals having an alteration in both alleles of the CD163 gene.
19. The method of claim 18 , where the animals having an alteration in both alleles of the CD163 gene exhibit increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV).