Methods for improving the health of porcine species by targeted inactivation of CD163
The present disclosure relates methods and compositions useful for prevention of porcine reproductive and respiratory syndrome virus (PRRSv) in animals, including animals of the species Sus scrofa . The present teachings relate to swine wherein at least one allele of a CD163 gene has been inactivated, and to specific methods and nucleic acid sequences used in gene editing to inactivate the CD163 gene. Swine wherein both alleles of the CD163 gene are inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSv). Elite lines comprising homozygous CD163 edited genes retain their superior properties.
1. A genetically modified pig whose genome comprises a CD163 gene comprising the nucleic acid sequence of SEQ ID NO: 453, wherein the pig expresses CD163 protein that exhibits reduced binding to porcine reproductive and respiratory syndrome virus (PRRSV) relative to a wild-type CD163 protein.
2. A cell isolated from the genetically modified pig according to claim 1 .
3. An isolated cell line obtained from the genetically modified pig of claim 1 .
4. An isolated fibroblast cell line obtained from the genetically modified pig of claim 1 .
5. The genetically modified pig of claim 1 , wherein the genetically modified pig is heterozygous for the nucleic acid sequence of SEQ ID NO: 453.
6. A cell isolated from the genetically modified pig according to claim 5 .
7. An isolated cell line obtained from the genetically modified pig of claim 5 .
8. An isolated fibroblast cell line obtained from the genetically modified pig of claim 5 .
9. The genetically modified pig of claim 1 , wherein the genetically modified pig is homozygous for the nucleic acid sequence of SEQ ID NO: 453.
10. A cell isolated from the genetically modified pig according to claim 9 .
11. An isolated cell line obtained from the genetically modified pig of claim 9 .
12. An isolated fibroblast cell line obtained from the genetically modified pig of claim 9 .
13. A method of producing a genetically modified pig, the method comprising:
a) introducing:
i) a Cas9 protein or a nucleic acid sequence encoding a Cas9 protein;
ii) a guide RNA (gRNA) comprising the nucleic acid sequence of SEQ ID NO: 249; and
iii) a gRNA comprising the nucleic acid sequence of SEQ ID NO: 256 into an isolated pig cell;
b producing a genetically modified pig from the isolated pig cell, wherein the genetically modified pig comprises at least some cells whose genomes comprise the nucleic acid sequence of SEQ ID NO: 453 and express, CD163 protein that exhibits reduced binding to porcine reproductive and respiratory syndrome virus (PRRSV) relative to a wild-type CD163 protein.
14. The method of claim 13 , wherein the isolated pig cell is a fertilized oocyte, and further wherein the producing comprises implanting the fertilized oocyte into a surrogate mother.
15. The method according to claim 13 , further comprising breeding the genetically modified pig to a wild-type pig to create a plurality of first generation progeny.
16. The method according to claim 15 , further comprising:
breeding the plurality of first generation progeny to produce a plurality of second generation progeny.
17. The method according to claim 16 , wherein individuals from the plurality of second progeny are homozygous for the nucleic acid sequence of SEQ ID NO: 453.
18. The method according to claim 13 , wherein the isolated pig cell is a fibroblast cell.
19. A genetically modified pig produced by the method of claim 13 .
20. The method according to claim 13 , wherein the producing somatic cell nuclear transfer of the isolated pig cell or a cell derived therefrom.
21. The method according to claim 20 , further comprising implanting the pig embryo obtained from the somatic cell nuclear transfer into a surrogate mother.
22. The method according to of claim 13 , wherein the isolated pig cell is a pig zygote or embryo.
23. The method according to of claim 22 , wherein the producing comprises implanting the pig zygote or embryo into a surrogate mother.