Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.

Keywords:

• CD163/exon 7
• CRISPR/Cas9
• disease resistance
• gene editing
• porcine reproductive and respiratory syndrome virus

Acknowledgments

The authors express thanks to Professor Wen-Bin Chung, National Pingtung University of Science and Technology, for kindly providing PRRSV 763; Ms. Ying-Ching Hung for her experimental assistance in the study of in vitro PRRSV challenge; Ms. J.-H. Chang for her assistance in porcine surgical operation and embryo transfer and Mrs. Chin-Liang Li, Shih-Chung Wang and Yuan-Tzu Wang for their assistance with pig management and KO pig care.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This study [Grant MOST 107-2622-B-866-001-CC2] was supported by the Ministry of Science and Technology, Executive Yung, and Fa Chang Enterprise Co. Ltd., Taiwan, ROC.