Pig with a genetically modified CD163 gene resistant to PRRSv
Inventors
Cigan, Andrew Mark • Lightner, Jonathan Edward • Culbertson, Matthew Scott • Christianson, William Thomas • Beaton, Benjamin • Burger, Brian • Barnes, Dylan • Campbell, Matthew
Assignees
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Publication Number
US-11208659-B2
Publication Date
2021-12-28
Expiration Date
Abstract
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.
Core Innovation
The disclosed subject matter relates to genetically modified Sus scrofa in which an endogenous CD163 gene is genetically modified to comprise a homozygous deletion of exon 7. The genetically modified CD163 gene comprises the nucleic acid sequence of SEQ ID NO: 453, and the resulting Sus scrofa is resistant to porcine reproductive and respiratory syndrome (PRRSV).
The disclosure further provides a defined pair of guide RNAs (gRNAs) for editing a Sus scrofa CD163 gene. The guide sequences are specified as the nucleic acid sequence of SEQ ID NO: 249 and the nucleic acid sequence of SEQ ID NO: 256, respectively, and are used together with Cas9 in a method of making a Sus scrofa that is resistant to PRRSV.
A nucleic acid sequence encoding Sus scrofa CD163 with a deletion in exon 7 is also provided. The nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 453 and is capable of conferring resistance to PRRSV in Sus scrofa, including embodiments in isolated cells and cell lines.
Claims Coverage
The independent claims cover four inventive features centered on PRRSV resistance mediated by a homozygous exon 7 deletion in the endogenous CD163 gene and a CD163 nucleic acid defined by SEQ ID NO: 453, together with a specific pair of gRNAs and a method of making the resistant Sus scrofa.
Homozygous exon 7 deletion in endogenous CD163 with SEQ ID NO: 453 for PRRSV resistance
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).
Isolated nucleic acid encoding CD163 exon 7 deletion (SEQ ID NO: 453) for PRRSV resistance
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.
Pair of gRNAs for CD163 editing defined by SEQ ID NO: 249 and SEQ ID NO: 256
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.
Making PRRSV-resistant Sus scrofa by Cas9 and gRNAs (SEQ ID NO: 249 and 256) producing a homozygous exon 7 CD163 deletion
A method of making a Sus scrofa that is resistant to porcine reproductive and respiratory syndrome virus (PRRSV), the method comprising 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; and implanting the zygote into a recipient female such that a Sus scrofa is obtained from the implanted zygote.
Coverage centers on PRRSV resistance mediated by a homozygous exon 7 deletion in the endogenous CD163 gene, the associated CD163 sequence defined by SEQ ID NO: 453, a specific pair of gRNAs defined by SEQ ID NO: 249 and SEQ ID NO: 256, and a zygote-based method using Cas9 and implantation to obtain the resistant Sus scrofa.
Stated Advantages
Resistance to porcine reproductive and respiratory syndrome (PRRSV).
Nucleic acid capable of conferring PRRSV resistance in Sus scrofa.
Provides Sus scrofa that is resistant to porcine reproductive and respiratory syndrome (PRRSV).
Documented Applications
Production of PRRSV-resistant Sus scrofa by generating a homozygous deletion of exon 7 in an endogenous CD163 gene with CD163 nucleic acid sequence of SEQ ID NO: 453.
Genetic modification of Sus scrofa to confer resistance to PRRSV via a homozygous deletion of exon 7 in endogenous CD163 with a CD163 nucleic acid sequence of SEQ ID NO: 453.
Editing of a Sus scrofa CD163 gene using a pair of gRNAs defined by SEQ ID NO: 249 and SEQ ID NO: 256 for generation of PRRSV-resistant Sus scrofa.
Use of an isolated nucleic acid sequence encoding Sus scrofa CD163 exon 7 deletion (SEQ ID NO: 453) that is capable of conferring resistance to PRRSV in Sus scrofa.
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