Pig with a genetically modified CD163 gene resistant to PRRSv
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 transgenic Sus scrofa whose genome comprises a homozygous deletion of exon 7 of an endogenous CD163 gene, wherein the genetically modified CD163 gene comprises the nucleic acid sequence of SEQ ID NO: 453, and wherein the Sus scrofa is resistant to porcine reproductive and respiratory syndrome (PRRSV).
2. The genetically modified Sus scrofa of claim 1 , wherein the Sus scrofa has the genome of a cell line deposited under ATCC accession number PTA-125814, PTA-125815, PTA-125816, PTA-125811, PTA-125907, PTA-125812, or PTA-125813.
3. A cell isolated from the Sus scrofa of claim 1 .
4. An isolated cell line obtained from the Sus scrofa of claim 1 .
5. An isolated fibroblast cell line obtained from the Sus scrofa of claim 1 .
6. An isolated nucleic acid sequence encoding Sus scrofa CD163 with a deletion in exon 7, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 453 and is capable of conferring resistance to porcine reproductive and respiratory syndrome (PRRSV) in Sus scrofa.
7. An isolated cell whose genome comprises the nucleic acid sequence of claim 6 .
8. A cell line comprising a plurality of the isolated cell of claim 7 .
9. The cell line of claim 8 , wherein the isolated cell is a fibroblast.
10. The isolated cell of claim 7 , wherein the cell is deposited under ATCC accession number PTA-125814, PTA-125815, PTA-125816, PTA-125811, PTA-125907, PTA-125812, or PTA-125813.
11. A pair of guide RNAs (gRNAs) for editing a Sus scrofa CD163 gene comprising:
a gRNA comprising a guide sequence consisting of the nucleic acid sequence of SEQ ID NO: 249; and
a gRNA comprising a guide sequence consisting of the nucleic acid sequence of SEQ ID NO: 256.
12. A method of making a Sus scrofa that is resistant to porcine reproductive and respiratory syndrome virus (PRRSV), the method comprising:
a) introducing Cas9 and a pair of gRNAs consisting of the nucleic acid sequences of SEQ ID NO: 249 and 256 into an isolated Sus scrofa zygote such that a zygote is created whose genome comprises a homozygous deletion of exon 7 of an endogenous CD163 gene, wherein the CD163 gene comprises the nucleic acid sequence of SEQ ID NO: 453;
b) implanting the zygote obtained in step a) into a recipient female such that a Sus scrofa is obtained from the implanted zygote, wherein the Sus scrofa obtained from the implanting has a genome comprising a genetically modified homozygous deletion of exon 7 of an endogenous CD163 gene, has a CD163 gene comprising the nucleic acid sequence of SEQ ID NO: 453, and is resistant to PRRSV.
13. The method of claim 12 , wherein the method comprises administering Cas9, a gRNA comprising a guide sequence consisting of the nucleic acid sequence of SEQ ID NO: 249; and a gRNA comprising a guide sequence consisting of the nucleic acid sequence of SEQ ID NO: 256.
14. The method according to claim 12 , wherein the Cas9 and the gRNAs are introduced as a pre-formed ribonucleoprotein (RNP) complex.
15. The method of claim 12 , wherein the Sus scrofa obtained from the implanting has the genome of a cell line deposited under ATCC accession number PTA-125814, PTA-125815, PTA-125816, PTA-125811, PTA-125907, PTA-125812, or PTA-125813.