Extensive germline genome engineering in pigs


The clinical applicability of porcine xenotransplantation—a long-investigated alternative to the scarce availability of human organs for patients with organ failure—is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR–Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs’ immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

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Fig. 1: The engineering of PERVKO·3KO·9TG pigs.
Fig. 2: PERVKO·3KO·9TG pig engineering and validation of the 3KO and 9TG edits at the genomic level.
Fig. 3: Validation of 3KO and 9TG in 3KO·9TG pigs and PERVKO·3KO·9TG pigs at the mRNA and protein levels.
Fig. 4: Functional validation of PERVKO·3KO·9TG pigs in mitigating human antibody binding, complement toxicity and NK cell toxicity, and modulating coagulation function.

Data availability

The main data supporting the findings of this study are available within the paper and its Supplementary Information. Data from the RNA-seq analyses are available at figshare (https://doi.org/10.6084/m9.figshare.12841418.v1). The raw data generated during the study are available at the China National GeneBank, with the accession code CNP0001254. The pig reference genome (Sus scrofa 11.1) sequence was obtained from Ensembl (ftp://ftp.ensembl.org/pub/release-91/fasta/sus_scrofa/dna). The pig transcript isoform information was obtained from the APPRIS database (http://appris.bioinfo.cnio.es/#/seeker).


  1. 1.

    Sykes, M. & Sachs, D. H. Transplanting organs from pigs to humans. Sci. Immunol. 4, eaau6298 (2019).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Cooper, D. K. C., Ekser, B. & Tector, A. J. Immunobiological barriers to xenotransplantation. Int. J. Surg. 23, 211–216 (2015).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Denner, J. & Tonjes, R. R. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin. Microbiol Rev. 25, 318–343 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Patience, C., Takeuchi, Y. & Weiss, R. A. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282–286 (1997).

    CAS  PubMed  Google Scholar 

  5. 5.

    Shin, J. S. et al. Minimizing immunosuppression in islet xenotransplantation. Immunotherapy 6, 419–430 (2014).

    CAS  PubMed  Google Scholar 

  6. 6.

    Niu, D. et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357, 1303–1307 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Cooper, D. K. Modifying the sugar icing on the transplantation cake. Glycobiology 26, 571–581 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Byrne, G., Ahmad-Villiers, S., Du, Z. & McGregor, C. B4GALNT2 and xenotransplantation: a newly appreciated xenogeneic antigen. Xenotransplantation 25, e12394 (2018).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Song, K. H. et al. Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem. J. 427, 179–188 (2010).

    CAS  PubMed  Google Scholar 

  10. 10.

    Estrada, J. L. et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation 22, 194–202 (2015).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Phelps, C. J. et al. Production of α1,3-galactosyltransferase-deficient pigs. Science 299, 411–414 (2003).

    CAS  PubMed  Google Scholar 

  12. 12.

    Lai, L. et al. Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092 (2002).

    CAS  PubMed  Google Scholar 

  13. 13.

    Martens, G. R. et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation 101, e86–e92 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Yamada, K. et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of α1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat. Med. 11, 32–34 (2005).

    CAS  PubMed  Google Scholar 

  15. 15.

    Kuwaki, K. et al. Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat. Med. 11, 29–31 (2005).

    CAS  PubMed  Google Scholar 

  16. 16.

    Cooper, D. K., Ekser, B., Ramsoondar, J., Phelps, C. & Ayares, D. The role of genetically engineered pigs in xenotransplantation research. J. Pathol. 238, 288–299 (2016).

    PubMed  Google Scholar 

  17. 17.

    Mohiuddin, M. M. et al. B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months. Am. J. Transpl. 12, 763–771 (2012).

    CAS  Google Scholar 

  18. 18.

    Zhou, C. Y. et al. Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation 12, 142–148 (2005).

    PubMed  Google Scholar 

  19. 19.

    Griesemer, A., Yamada, K. & Sykes, M. Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol. Rev. 258, 241–258 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Lilienfeld, B. G., Crew, M. D., Forte, P., Baumann, B. C. & Seebach, J. D. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation 14, 126–134 (2007).

    PubMed  Google Scholar 

  21. 21.

    Ide, K. et al. Role for CD47-SIRPα signaling in xenograft rejection by macrophages. Proc. Natl Acad. Sci. USA 104, 5062–5066 (2007).

    CAS  PubMed  Google Scholar 

  22. 22.

    Siegel, J. B. et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation 64, 888–896 (1997).

    CAS  PubMed  Google Scholar 

  23. 23.

    Lee, K. F. et al. Recombinant pig TFPI efficiently regulates human tissue factor pathways. Xenotransplantation 15, 191–197 (2008).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Choi, C. Y. et al. Pig tissue factor pathway inhibitor α fusion immunoglobulin inhibits pig tissue factor activity in human plasma moderately more efficiently than the human counterpart. Biotechnol. Lett. 39, 1631–1638 (2017).

    CAS  PubMed  Google Scholar 

  25. 25.

    Robson, S. C., Cooper, D. K. & d’Apice, A. J. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation 7, 166–176 (2000).

    CAS  PubMed  Google Scholar 

  26. 26.

    Ji, H. et al. Pig BMSCs transfected with human TFPI combat species incompatibility and regulate the human TF pathway in vitro and in a rodent model. Cell. Physiol. Biochem. 36, 233–249 (2015).

    PubMed  Google Scholar 

  27. 27.

    Kopp, C. W. et al. Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors. Transplantation 63, 749–758 (1997).

    CAS  PubMed  Google Scholar 

  28. 28.

    Iwase, H., Ezzelarab, M. B., Ekser, B. & Cooper, D. K. The role of platelets in coagulation dysfunction in xenotransplantation, and therapeutic options. Xenotransplantation 21, 201–220 (2014).

    PubMed  Google Scholar 

  29. 29.

    Miwa, Y. et al. Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation. Xenotransplantation 17, 26–37 (2010).

    PubMed  Google Scholar 

  30. 30.

    Mohiuddin, M. M. et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat. Commun. 7, 11138 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Wheeler, D. G. et al. Transgenic swine: expression of human CD39 protects against myocardial injury. J. Mol. Cell Cardiol. 52, 958–961 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Cooper, D. K. C. et al. Justification of specific genetic modifications in pigs for clinical organ xenotransplantation. Xenotransplantation 26, e12516 (2019).

    PubMed  Google Scholar 

  33. 33.

    Samy, K. P., Martin, B. M., Turgeon, N. A. & Kirk, A. D. Islet cell xenotransplantation: a serious look toward the clinic. Xenotransplantation 21, 221–229 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Matsumoto, S., Tomiya, M. & Sawamoto, O. Current status and future of clinical islet xenotransplantation. J. Diabetes 8, 483–493 (2016).

    PubMed  Google Scholar 

  35. 35.

    Fischer, K. et al. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Sci. Rep. 6, 29081 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Yunga, G. L. P., Riebenb, R., Bühlerc, L., Schuurmanc, H. J. & Seebach, J. Xenotransplantation: where do we stand in 2016? Swiss Med. Wkly 147, w14403 (2017).

    Google Scholar 

  37. 37.

    Langin, M. et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature 564, 430–433 (2018).

    PubMed  Google Scholar 

  38. 38.

    Kim, S. C. et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion. Am. J. Transpl. 19, 2174–2185 (2019).

    CAS  Google Scholar 

  39. 39.

    Li, X. et al. PiggyBac transposase tools for genome engineering. Proc. Natl Acad. Sci. USA 110, E2279–E2287 (2013).

    CAS  PubMed  Google Scholar 

  40. 40.

    Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Laird, C. T. et al. Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury. Xenotransplantation 24, e12294 (2017).

    Google Scholar 

  43. 43.

    Chen, D. et al. Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins. Transplantation 68, 832–839 (1999).

    CAS  PubMed  Google Scholar 

  44. 44.

    Fischer, K. et al. Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation 27, e12560 (2020).

    PubMed  Google Scholar 

  45. 45.

    Ekser, B., Markmann, J. F. & Tector, A. J. Current status of pig liver xenotransplantation. Int. J. Surg. 23, 240–246 (2015).

    PubMed  Google Scholar 

  46. 46.

    Zhao, Y. et al. Skin graft tolerance across a discordant xenogeneic barrier. Nat. Med. 2, 1211–1216 (1996).

    CAS  PubMed  Google Scholar 

  47. 47.

    Jesus, B. B. D. et al. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. EMBO Mol. Med. 4, 691–704 (2012).

    Google Scholar 

  48. 48.

    Kennedy, E. M. & Cullen, B. R. Gene editing: a new tool for viral disease. Annu Rev. Med 68, 401–411 (2017).

    CAS  PubMed  Google Scholar 

  49. 49.

    Burkard, C. et al. Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection. J. Virol. 92, e00415-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Yan, Q. et al. Production of transgenic pigs over-expressing the antiviral gene Mx1. Cell Regen. 3, 11 (2014).

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Yang, L. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101–1104 (2015).

    CAS  PubMed  Google Scholar 

  52. 52.

    Wei, H. et al. Comparison of the efficiency of banna miniature inbred pig somatic cell nuclear transfer among different donor cells. PLoS ONE 8, e57728 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Tomii, R. et al. Production of cloned pigs by nuclear transfer of preadipocytes following cell cycle synchronization by differentiation induction. J. Reprod. Dev. 55, 121–127 (2009).

    PubMed  Google Scholar 

  54. 54.

    Kurome, M. et al. Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer. J. Reprod. Dev. 54, 254–258 (2008).

    PubMed  Google Scholar 

  55. 55.

    Costa, C. & Manez, R. Xenotransplantation: Methods and Protocols 335 (Springer, xiHumana Press, 2012).

  56. 56.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    McKenna, A. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Zhu, L. J., Holmes, B. R., Aronin, N. & Brodsky, M. H. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS ONE 9, e108424 (2014).

    PubMed  PubMed Central  Google Scholar 

  60. 60.

    McLaren, W. et al. The ensembl variant effect predictor. Genome Biol. 17, 122 (2016).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Rodriguez, J. M. et al. APPRIS: annotation of principal and alternative splice isoforms. Nucleic Acids Res. 41, D110–D117 (2013).

    CAS  PubMed  Google Scholar 

  62. 62.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  63. 63.

    Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank G. Yang of Harvard University for reading our manuscript; Q. Tang and P. O’Connell for their advice; and Y. Yang and Q. Yang from Third Affiliated Hospital of Sun Yat-sen University, H. Liu from Henan Chuangyuan Biotechnology, and colleagues at Qihan Bio and eGenesis for their technical assistance and discussions. The pig cloning work was supported by National Key R&D Program of China (grant no. 2019YFA0110700).

Author information




L.Y., G.M.C. and Y.G. envisioned and supervised the whole project; H.-J.W. and H.-Y.Z. supervised pig cloning and production. Y.Y., W.X. and Y.K. designed the experiments and wrote the manuscript. Y.Y., Y.K., Y.Z., X.S., L.Lamriben, J.W., J.X., M.X., Q.Z., Y.L., J.V.L., M.L., V.P., M.E.Y., Z.S., Y.D., W.W., H.D., L.S., X.W., L.Le, X.F, H.G., R.A. and S.Y.W. performed experiments. W.X., D.G., M.Y. and M.G. analysed the data. J.G., S.M., D.J., T.D.N. and Z.L. performed pig cloning and generated pigs. J.M., W.Q. and W.F.W. revised the manuscript.

Corresponding author

Correspondence to Luhan Yang.

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Competing interests

Y.Y., W.X., Y.Z., X.S., M.Y., J.W., J.X., M.X., Q.Z., Y.L., H.D., L.S., X.W., L.Le, X.F., Y.G. and L.Y. are employed by Qihan Bio Inc. Y.K., D.G., L.Lamriben, J.V.L., M.L., V.P., M.E.Y., H.G., R.A., S.Y.W., W.F.W. and W.Q. are employed by eGenesis Inc. M.G. is a consultant to Qihan Bio Inc. and eGenesis Inc. J.M. is an advisor on the scientific advisory board of Qihan Bio Inc. and eGenesis Inc. G.M.C. is the cofounder and scientific advisor of Qihan Bio Inc. and eGenesis Inc. Y.K., M.G., W.Q., Y.G. and L.Y. are listed as inventors on a provisional patent application pertaining to the results of the paper.

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Yue, Y., Xu, W., Kan, Y. et al. Extensive germline genome engineering in pigs. Nat Biomed Eng (2020). https://doi.org/10.1038/s41551-020-00613-9

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