FFTC Agricultural Policy Platform (FFTC-AP)

ABSTRACT

In recent years, gene editing (GE) techniques, such as CRISPR/Cas9, are broadly applied to agricultural and medical sectors because of the characters of low technical barriers and high efficiency. In this review, we will concentrate on using GE as a precise new breeding technique (NBT) in the pig industry, including in the fields of production, disease resistance, and biomedical usage. The relative regulatory cost barriers will also be discussed. We hope the regulation and benefit debate of GE could be cleared as a base on science and case-by-case principle and the NBT achievements could be realized for practical usage, including food chain.

Keywords: CRISPR/Cas9, gene-editing (GE), new breeding technique (NBT), pig, regulation.

INTRODUCTION

The genetic diversity of natural life is an evolutional result on earth to accommodate for survival in their natural environments. In the research field of crops, in vitro mutagenesis has been used for breeding and increasing the diversity of genetic background; on the other hand, the superior livestock got more opportunities to transmit genetic sources to their offspring, but that also caused reduction in genetic diversity. Although in swine the wild species survive because of environmental factors, the commercial breeds are selected according to the performance of phenotype as dominant factors, and currently, pig breeds could be designed by new breeding techniques (NBT) (Zhao et al., 2019). However, in the progressed crucial environment, the wild species might retain their unique ability to survive, e.g., tolerance to African swine fever (ASF) infection in warthog (cf. Penrith et al., 2021) and heat tolerance in cattle (Hansen, 2020). Currently, whole-genome sequencing reveals many valuable genetic sources and offers the opportunity to use that information for improving the traits of commercial breeds by NBT.

The technologies of genome/gene editing (GE) include ZFN (zinc finger nuclease) (Moehle et al., 2007), TALEN (transcription activator-like effector nuclease) (Christian et al., 2010), and CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated (Cas) endoribonuclease 9) (Cong et al., 2013). CRISPR/Cas9 is a major methodology as precise NBT with high efficiency, low technical barrier, low cost, and can be broadly applied for various organisms. CRISPR/Cas9 uses a single-guide RNA (sgRNA) to distinguish target DNA sequence in a Watson-Crick base pairing way, which had been developed in early 2012 (Jinek et al., 2012), and selectively cleaves the target DNA (Josephs et al., 2015) and results in target gene mutation and generation of new variants. These NBTs could be valuably applied for agricultural and medical purposes. Moreover, since transiently expressing vectors are used, none of them will be left in the target organisms without carrying any exogenes or transgenes. It is worth noting that the mutants created by NBTs cannot be distinguished from their natural counterparts. Therefore, such kinds of mutants could be recognized as natural variants. In the following, we will briefly review the global trends of GE regulation and the application or progress of GE on pig industry and medical purposes.

THE GLOBAL TRENDS OF REGULATION ON NEW BREEDING TECHNIQUE

The NBT of GEs, especially CRISPR/Cas9, have high efficiency and fast achievement, low developing cost and technique barrier, and broad application to many organisms. However, the created variants might carry many types of indels and off-targeting mosaicism. The organisms or products made by GE are still debated as genetically modified organisms (GMO) and regulated in diverse ways. Based on carrying exo-nuclear acid or not, Argentina, Austria, Brazil, Canada, Chile, and Japan have exempted those organisms without exo-DNA as a non-GMO (Schmidt et al., 2020; Entine et al., 2021). Especially in Japan, being a signatory country, according to the definition of Cartagena Protocol on Biosafety, the "Living modified organism” means any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology, which exempted the GE products without carrying exogene from GMO regulation. The USA has revised the Guidance for Industry 187 to “Regulation of Intentionally Altered Genomic DNA in Animals (2017 draft),” all GE products (e.g., milk, meat, and eggs) are unsalable before passing the application of the new animal drug (NADA). Furthermore, the European Court of Justice (ECJ) issued a rule in 2018 to state that organisms obtained by directed mutagenesis techniques are regarded as GMOs because their genome had been altered (according to process-based principle).

However, a recent positive trend and exciting study from the EU found that current GMO legislation is not fit for the new genome technique (NGT) that alters the genome of organisms (Byrne, 2021). The study claimed that several plant products obtained from NGT contribute to the objectives of the EU’s Green Deal and biodiversity strategies and even meet the United Nations’ sustainable development goals (SDGs) for a more resilient and sustainable agri-food system. Accordingly, current GE or NGT (or NBT) could be applied to domestic animals to achieve the same purposes and other fittings for animal welfare.  

Recently, the FDA has approved the GGTA1 gene-edited or KO pigs (alias GalSafe pigs) to enter the pork consumption market (FDA, 2020) and progressing the premarketing review of the porcine reproductive and respiratory syndrome virus (PRRSV) resistance pigs. The regulatory barriers decrease the developing opportunity and delay the market to be enlarged. As an example, the PRRSV resistance pig loses more than US$28.3 billion in the USA and EU markets if it's delayed to the market for 15 years (Van Enennaam et al., 2020). The regulatory barriers also block the small-scale research teams or companies from developing and promoting their research accomplishments to practical usage. If following the regulations of GMOs, the developing country would be difficult to gain the benefits of scientific development to improve their food supply. Since among the NBT’s broad applications, pigs' growth, health, and nutrients are in the normal range, based on scientific and case by case principle, the GE pigs and their products could be exempted from regulating as GMO or GM products. Those may be quickly allowed to launch on the market as the previously mentioned "Galsafe pigs."

APPLICATIONS OF GENE-EDITING TECHNIQUE IN THE PIG INDUSTRY

Application of new breeding technology on pig production

The precise NBT has been applied in pig production fields in past years, including pigs’ growth, meat production, disease resistance, and piglet warming. In pig growth, the myostatin KO Meishan pigs had been first generated by ZFN and shown to increase the meat growth with double muscling on the hip similar to that in cattle (Grobet et al., 1997, 1998). These homozygotic KO pigs were grown healthily to adults (Qian et al., 2015). However, only native breeds, e.g., Erhualian KO pigs (Wang et al., 2017) and Liang Guang Small Spotted pigs (Li et al., 2020), could repeat the achievement, but not in those commercial breeds, including Landrace and Large White (Wang et al., 2015 and 2016) and Ducro (Zou et al., 2019). Since then, Wang et al. (2017) doubted that commercial breeds might be more sensitive to KO endogenous genes. After that, by using CRISPR/Cas9, Xiang et al. (2018) mutated IGF2 intron 3–3072 site of IGF2 gene and abolished repressor ZBED6 binding improved meat production in Bama minipigs, and Zou et al. (2018) generated FBOX 40 KO pigs which increased IRS1 expression and muscle growth in Chinese experimental minipig. Evenly, Zhu et al. (2020) had found the cloned IRX3-/- Bama minipigs revealed that the surviving IRX3-/- KO piglets were significantly lower in terms of birth weight, poor viability, and all piglets die shortly after birth. Although the impacts on viability might be caused by somatic cell nuclear transfer, the authors suggested that IRX3 may have essential biological functions and could not be KO to reduce fat content (Zhu et al., 2020).

The FDA has permitted the GGTA1 gene-edited or KO pigs to release on the market as GalSafe pigs (FDA, 2020), which the α-Gal being eliminated from pork and people with α-Gal syndrome could avoid a severe allergic reaction to the α-Gal, which is also found in beef and lamb. The KO pig is the first case of an animal biotechnology product that has been launched for food and biomedical usages. In red meat, it also expresses tremendous N-glycolylneuraminic acid (Neu5Gc; non-human glycan) (Jahan et al., 2021), which could increase the happening of colorectal cancer and Atherosclerosis (Alisson-Silva et al., 2016). We have generated alpha-gal (Chuang et al., 2017) and Neu5Gc (Tu et al., 2019) KO pigs by CRISPR/Cas9 GE; and proven that intestinal decellular scaffold (extra-cellular matrix, ECM) from the dKO pigs could evoke less inflammation than from wild type (WT) pigs after being implanted into the dKO pigs (Yen et al., 2020). Accordingly, we suggest that dKO pigs will offer good pork as healthy red meat than WT pigs.

Application of new breeding technology on pig disease resistance

The PRRSV emerged in the late 1980s and rapidly became one of the most significant viral pathogens in the pig industry. Due to no efficacy vaccines developed, it was estimated to have caused a US$660 million loss in the USA and two piglets lose per sow in Taiwan per year. In vivo, the virus shows a very narrow cell tropism and targets specific subsets of porcine monocytes/macrophages and infects the cells via heparan sulfate, sialoadhesin (CD169), and CD163 as receptors (Van Breedam et al., 2010). Currently, the most convincing receptor is CD163 on porcine alveolar macrophages (pAM) as infective receptors of PRRSV (reviewed by Zhang and Yoo, 2015). The manipulation of the CD163 gene, including KO (Whitworth et al., 2016; Yang et al., 2018; Tanihara et al., 2021; Xu et al., 2020), to delete the exon 7 of CD163 protein (Burkard et al., 2017 and 2018; Wang, H. et al., 2019), and even to delete part of exon 7 on the infective pocket of virons (Guo et al., 2019). All these GE pigs can fully resist PRRSV infection without disturbing the pigs' normal growth and health. Furthermore, Xu et al. (2020) had generated double KO (dKO), including KO CD163 and pAPN (porcine aminopeptidase N) pigs, which also being proven resistance to PRRSV and TGEV infection, respectively.

There are four genera of the coronavirus (CoVs), including α-, β-, γ-, and δ-coronavirus, identified in the past years. Six CoVs had been found having the ability to infect pigs, including four α-coronaviruses [(transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine epidemic diarrhea virus (PEDV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV)], one β-coronavirus [porcine hemagglutinating encephalomyelitis virus (PHEV)], and one porcine δ-coronavirus (PDCoV) (cf. Wang, Q. et al., 2019). Currently, TGEV, PRCV, and PHEV have caused little clinical effect, whereas PEDV, PDCoV, and SADS-CoV have caused severe acute diarrhea with high mortality and are considered emerging CoVs. It suggested that these viruses use S-protein to infect the host cells by contacting the pAPN and Neu5Gc. However, using CRISPR/Cas9 GE, neither pAPN (Whitworth et al., 2019) nor CMAH (to null express Neu5Gc; Tu et al., 2019) KO piglets could resist PEDV infection, which elucidated that both of them might not be the sufficient putative receptors of PEDV. But, pAPN KO pigs had been proven resistant to TGEV infection (Whiteworth et al., 2019; Xu et al., 2020) and decreased susceptibility to PDCoV infection (Xu et al., 2020).

In ASFV, the pAM had been proven the target cells (Alcami et al., 1990). The antibodies of CD163, the putative receptor, could inhibit both ASFV infection and viral particle binding to pAM (Sanchez-Torres et al., 2003). However, the CD163 KO pigs failed to resist the virus infection (Popescu et al., 2017). The Edinburgh group identified polymorphic variation in p65 and revealed 3 AA of p65 differences between a warthog and domestic pig (Palgrave et al., 2011). Furthermore, they had substituted the 3 AA from domestic pigs to warthog (Lillico et al., 2016) by using CRISPR/Cas9; but these edited pigs failed to confer resilience to ASFV (McCleary et al., 2020). In the German group, the transfected wild boar lung cell lines with a plasmid encoding Cas9 and a guide RNA target to the p30 gene of ASFV had been proven, which can completely abrogate the plaque formation ASFV and reduce the virus-free particles yields (Hübner et al., 2018). But in this approach, the Cas9 and guide RNA plasmid needs to be integrated into the genome of cells, and it might be debated as a GMO.

Recently, under the CRISPR/Cas9-mediated knock-in strategy, Xie et al. (2018) have generated transgenic (Tg) pigs carrying antiviral small hairpin RNAs (shRNAs) of anti-classical swine fever virus (CSFV) and demonstrated that these pigs could effectively limit the CSFV replication and reduce associated clinical signs and mortality. Furthermore, Hu et al. (2015) showed that Tg pigs constitutively expressed FMDV-specific shRNAs could resist the FMD virus by the Tg technique. The ASFV and CoVs research revealed that it's challenging to develop an effective vaccine and necessary to find a new strategy to enhance the biosecurity for pig production. As mentioned above, we suggest that combining the GE method and shRNAs procedure might be a practical approach. Directly targeted and degraded the genome of infected viruses might result in good resistance or significantly reduce viral infection.

Improving piglets’ survival in cold regions

Pigs have no brown adipose tissue and uncoupling protein 1 (UCP1), which is located in the inner plasma membrane of mitochondria and could block electron transition to formation ATP and emitted as the heat of non-shivering thermogenesis to keep body warming (Hou et al., 2017; Trayhurn et al., 1989). The exons 3 to 5 of the pig's CUP1 gene had been evolutionally deleted 20 million years ago (Berg et al., 2006), and pigs lack non-shivering thermogenesis, which caused the nursing piglets to need plenty of electric energy supplement for warming their body and survive, especially in cold regions and even in cool season. Zhang et al. (2017) constructed a porcine adiponectin promotor with mouse UCP1 cDNA and knock-in (KI) it at porcine UCP1 exon two sites through GE by CRISPR/Cas9. They concluded that KI piglets are more tolerant and survive cold stress, save economic energy loss, and improve animal welfare.

APPLICATIONS OF GENE-EDITED PIGS ON MEDICAL PURPOSES

For several years, GE pigs have been used to develop therapeutics, human disease models, medical devices, xenografts, and tailor-made human organs. The only therapeutics case was KI/KO pigs, which generated by KI human serum albumin (HSA) cDNA into porcine SA (PSA) gene (KO), and resulted in expressing only HSA without any PSA (Peng et al., 2015). Due to pigs’ anatomy, physiology, immunology, metabolism, and genetics are the most similar to human beings, except for non-human primates, and short reproductive cycle, the porcine animal models established by NBT had been developed for neurodegenerative disease, cardiovascular disease, cancer, and immunodeficiency (Yang and Wu, 2018). Furthermore, GE pigs offer better source animals for medical devices than WT (Yen et al., 2020). Although many efforts to use Tg and GE methods and immunosuppers, xenografts, e.g., porcine corneal, and even immune privilege in the eyes, it is still not practiced in the clinical trial of xenotransplantation (Yoon et al., 2021). Snice Kobayashi et al. (2010) had used the pancreatogenesis-disabled (Pdx1 KO) mouse and blastocyst complementation (BC) technique to generate rat pancreas in mice, the functional lungs had been further generated by using the conditional BC method in the mouse-to-mouse model (Mori et al., 2019). The disabled organogenesis pigs, including pancreatogenesis, nephrogenesis, hepatogenesis, and vasculogenesis, could also be used for tailor-made organs for clinic purposes (Matsunari et al., 2020). However, there are many challenges, including interspecies chimeras and ethical issues (Morata Tarifa et al., 2020), and organ farming and risk of viral transmission (Aravalli, 2021) remain to be concerned.

CONCLUSION

The GE is an elegant technique that could broadly improve pig production, e.g., increasing disease tolerance or resistance, enhancing animal welfare, medical usages, etc. Currently, most countries exempted GE products from GMOs, but how to suitably regulate scientific achievement, especially in pig production, no more and no less might be a clever strategy for human beings. Furthermore, the GE is a good NBT methodology that contributes to agrobiodiversity and a more sustainable food system with more resilience to disease and climate change while ensuring affordable solutions for farmers and consumers.  

REFERENCES

Alcami, A., Carrascosa, A.L., & Vinuela, E. (1990). Interaction of African swine fever virus with macrophages. Virus Research, 17, 93–104. https://doi: 10.1016/0168-1702(90)90071-i.

Alisson-Silva, F., Kawanishi, K., & Varki, A. (2016). Human risk of diseases associated with red meat intake: Analysis of current theories and proposed role for metabolic incorporation of a non-human sialic acid. Molecular Aspects Medicine, 51, 16-30. https://doi: 10.1016/j.mam.2016.07.002.

Aravalli, R.N. (2021). Generating liver using blastocyst complementation-Opportunities and challenges. Xenotransplantation 28, e12668. https://doi: 10.1111/xen.12668.

Berg, F., Gustafson, U., & Andersson, L. (2006). The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: A genetic explanation for poor thermoregulation in piglets. PLoS Genetics, 2, e129. https://doi: 10.1371/journal.pgen.0020129.        

Burkard, C., Lillico, S.G., Reid, E., Jackson, B., Mileham, A.J., Ait-Ali, T., Whitelaw, C.B., & Archibald, A.L. (2017). Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathogens, 13, e1006206. https://DOI: 10.1371/journal.ppat.1006206

Burkard, C., Opriessnig, T., Mileham, A.J., Stadejek, T., Ait-Ali, T., Lillico, S.G., Whitelaw, C.B.A., & Archibald, A.L. (2018). Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection. Journal of Virology, 92, pii: e00415-18. https://doi: 10.1128/JVI.00415-18.

Byrne, J. (2021). New plant breeding techniques: EU Commission finds GMO legislation not fit for purpose. https://www.feednavigator.com/Article/2021/04/29/EU-consultation-process-on-legal-framework-for-NGTs-to-begin.

Christian, M., Cermak, T., Doyle, E.L., Schmidt, C., Zhang, F., Hummel, A., Bogdanove, A.J., & Voytas, D.F. (2010). Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 186, 757-761. https://doi:10.1534/genetics.110.120717

Chuang, C.-k, Chen, C.-H., Huang, C.-L., Lin, Y.-J., Su, Y.-H., Peng, S.-H., Lin, T.-Y., Tai, H.-C., Yang, T.-S., & Tu, C.-F. (2017). Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors. Animal Biotechnology, 28, 174-181. https:// doi:10.1080/10495398.2016.1246453

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., & Zhang, F.. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819-823. https://doi: 10.1126/science.1231143

Entine, J., Felipe, M.S.S., Groenewald, J.H/, Kershen, D.L., Lema, M., McHughen, A., Nepomuceno, A.L., Ohsawa, R., Ordonio, R.L., Parrott, W.A., Quemada, H., Ramage, C., Slamet-Loedin, I., Smyth, S.J., & Wray-Cahen, D. (2021). Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world. Transgenic Research 30, 551-584. https://doi: 10.1007/s11248-021-00257-8.

FDA. (2020). FDA Approves First-of-its-kind intentional genomic alteration in line of domestic pigs for both human food, potential therapeutic uses. https://www.fda.gov/news-events/press-announcements/fda-approves-first-its-kind-intentional-genomic-alteration-line-domestic-pigs-both-human-food.

Grobet, L., Martin, L. J., Poncelet, D., Pirottin, D., Brouwers, B., Riquet, J., Schoeberlein, A., Dunner, S., Ménissier, F., Massabanda, J., Fries, R., Hanset, R., & Georges, M. (1997). A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genetics, 17, 71–74. https://doi: 10.1038/ng 0997-71.

Grobet, L., Poncelet, D., Royo, L. J., Brouwers, B., Pirottin, D., Michaux, C., Ménissier, F., Zanotti, M., Dunner, S., & Georges, M. (1998). Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle. Mammalian Genome, 9, 210–213. https://doi: 10.1007/s003359900727

Guo, C., Wang, M., Zhu, Z., He, S., Liu, H., Liu, X., Shi, X., Tang, T., Yu, P., Zeng, J., Yang, L., Cao, Y., Chen, Y., Liu, X., & He, Z. (2019). Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain, which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection. Frontiers in Immunology, 10, 1846. https://doi.org/10.3389/fimmu.2019.01846.

Hansen, P.J. (2020). Prospects for gene introgression or gene editing as a strategy for reduction of the impact of heat stress on production and reproduction in cattle. Theriogenology, 154, 190-202. https://doi: 10.1016/j.theriogenology.2020.05.010.

Hou, L., Shi, J., Cao, L., Xu, G., Hu, C., & Wang, C. (2017). Pig has no uncoupling protein 1. Biochemical and Biophysical Research Communications, 487, 795-800. https://doi: 10.1016/j.bbrc.2017.04.118.

Hu, S., Qiao, J., Fu, Q., Chen, C., Ni, W., Wujiafu, S., Ma, S., Zhang, H., Sheng, J., Wang, P., Wang, D., Huang, J., Cao, L., & Ouyang, H. (2015). Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection. eLife, 4, e06951. https://doi:10.7554/eLife.06951.

Hübner, A., Petersen, B., Keil, G.M., Niemann, H., Mettenleiter, T.C., & Fuchs, W. (2018). Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L). Scientific Reports, 8, 1449. https://doi: 10.1038/s41598-018-19626-1.

Jahan, M., Thomsona, P.C., Wynna, P.C., & Wang, B. (2021). The non-human glycan, N-glycolylneuraminic acid (Neu5Gc), is not expressed in all organs and skeletal muscles of nine animal species. Food Chemistry, 343, 128439. https://doi.org/10.1016/j.foodchem.2020.128439

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., & Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337, 816-821. https://doi:10.1126/science.1225829.

Josephs, E.A., Kocak, D.D., Fitzgibbon, C.J., McMenemy, J., Gersbach, C.A., & Marszalek, P.E. (2015). Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Research. 43, 8924-8941. https://doi: 10.1093/nar/gkv892

Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yama-zaki, Y., Ibata, M., Sato, H., Lee, Y.S., Usui, J., Knisely, A.S., Hirabayashi, M., & Nakauchi, H. (2010). Generation of rat pancreas in mouse by interspecific blasto-cyst injection of pluripotent stem cells. Cell 142, 787-799. https://doi: 10.1016/j.cell.2010.07.039.

Li, R., Zeng, W., Ma, M., Wei, Z., Liu, H., Liu, X., Wang, M., Shi, X., Zeng, J., Yang, L., Mo, D., Liu, X., Chen, Y., & He, Z. (2020). Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs. Transgenic Research, 29, 149-163. https://doi: 10.1007/s11248-020-00188-w.

Lillico, S.G., Proudfoot, C., King, T.J., Tan, W., Zhang, L., Mardjuki, R., Paschon, D.E., Rebar, E.J., Urnov, F.D., Mileham, A.J., McLaren, D.G., & Whitelaw, C.B. (2016). Mammalian interspecies substitution of immune modulatory alleles by genome editing. Scientific Reports, 6, 21645. https://doi: 10.1038/srep21645.

Matsunari, H., Watanabe, M., Hasegawa, K., Uchikura, A., Nakano, K., Umeyama, K., Masaki, H., Hamanaka, S., Yamaguchi, T., Nagaya, M., Nishinakamura, R., Nakauchi, H., & Nagashima, H. (2020). Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation. Stem Cell Reports 14, 21-33. https://doi:10.1016/j.stemcr.2019.11.008.

McCleary, S., Strong, R., McCarthy, R.R., Edwards, J.C., Howes, E.L., Stevens, L.M., Sánchez-Cordón, P.J., Núñez, A., Watson, S., Mileham, A.J,, Lillico, S.G., Tait-Burkard, C., Proudfoot, C., Ballantyne, M., Whitelaw, C.B.A, Steinbach, F., & Crooke, H.R. (2020). Substitution of warthog NF-κB motifs into RELA of domestic pigs is not sufficient to confer resilience to African swine fever virus. Scientific Reports, 10, 8951. https://doi: 10.1038/s41598-020-65808-1

Moehle, E.A., Rock, J.M., Lee, Y.L., Jouvenot, Y., DeKelver, R.C., Gregory, P.D., Umov, F.D., & Holmes, M.C. (2007). Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proceeding National Academy Sciences USA, 104, 3055-3060. https://doi: 10.1073/pnas.0611478104

Morata Tarifa, C., López Navas, L., Azkona, G., & Sánchez Pernaute. R. (2020). Chimeras for the twenty-first century. Critical Reviews Biotechnology 40, 283-291. doi: 10.1080/07388551.2019.1679084

Mori, M., Furuhashi, K., Danielsson, J.A., Hirata, Y., Kakiuchi, M., Lin, C.S., Ohta, M., Riccio, P., Takahashi, Y., Xu, X., Emala, C.W., Lu, C., Nakauchi H, & Cardoso WV. (2019). Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells. Nature Medicine 25, 1691-1698. https://doi: 10.1038/s41591-019-0635-8.

Palgrave, C.J., Gilmour, L., Lowden, C.S., Lillico, S.G., Mellencamp, M.A., & Whitelaw, C.B. (2011). Species-specific variation in RELA underlies differences in NF-kappaB activity: a potential role in African swine fever pathogenesis. Journal of virology, 85, 6008–6014. https://doi: 10.1128/jvi.00331-11.

Peng, J., Wang, Y., Jiang, J., Zhou, X., Song, L., Wang, L., Ding, C., Qin, J., Liu, L., Wang, W., Liu, J., Huang, X., Wei, H., & Zhang, P. (2015). Production of human albumin in pigs through CRISPR-Cas9-mediated Knockin of human cDNA into swine albumin locus in the zygotes. Sci Rep. 5:16705.

Penrith, M.-L., Bastos, A., & Chenais, E. (2021). With or without a vaccine—A review of complementary and alternative approaches to managing african swine fever in resource-constrained smallholder settings. Vaccines, 9, 116. https://doi: 10.3390/vaccines9020116.

Popescu, L., Gaudreault, N. N., Whitworth, K. M., Murgia, M. V., Nietfeld, J. C., Mileham, A., Samuel, M., Wells, K. D., Prather, R. S., & Rowland R. R.R. (2017). Genetically edited pigs lacking CD163 show no resistance following infection with the ASFV isolate, Georgia 2007-1. Virology, 501, 102-106. https://DOI: 10.1016/j.virol.2016.11.012

Qian, L., Tang, M., Yang, J., Wang, Q., Cai, C., Jiang, S., Li, H., Jiang, K., Gao, P., Ma, D., Chen, Y., An, X., Li, K., & Cui, W. (2015). Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Scientific Reports, 5, 14435. https://doi: 10.1038/srep14435.

Sanchez-Torres, C., Gomez-Puertas, P., Gomez-del-Moral, M., Alonso, F., Escribano, J.M., Ezquerra, A., & Dominguez, J. (2003). Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection. Archives of Virology, 148, 2307–2323. https://doi: 10.1007/s00705-003-0188-4.

Schmidt, S.M., Belisle, M., & Frommer, W.F. (2020). The evolving landscape around genome editing in agriculture: Many countries have exempted or move to exempt forms of genome editing from GMO regulation of crop plants. EMBO Reports, 21, e50680. https://doi.org/10.15252/embr.202050680.

Tanihara, F., Hirata, M., Nguyen, N. T., Le, Q. A., Wittayarat, M., Fahrudin, M., Hirano, T., & Otoi, T. (2021). Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes. Animal Biotechnology, 32, 147-154. https://doi: 10.1080/10495398.2019.1668801

Trayhurn, P., Temple, N.J., & Van Aerde, J. (1989). Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig. Canadian Journal of Physiology and Pharmacology, 67, 1480–1485. https://doi.org/10.1139/y89-239

Tu, C.-F., Chuang, C.-K., Hsiao, K.-H., Chen, C.-H., Chen, C.-M., Peng, S.-H., Su, Y.-H., Chiou, M.-T., Yen, C.-H., Hung, S.-W., Yang, T.-S-, & Chen, C.-M. (2019). Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression. PLoS One, 14, e0217236. https://doi: 10.1371/journal.pone.0217236

Van Breedam, W., Delputte, P.L., Van Gorp, H., Misinzo, G., Vanderheijden, N., Duan, X., & Nauwynck, H.J. (2010). Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. Journal of General Virology, 91(Pt 7), 1659-67. https://doi: 10.1099/vir.0.020503-0.

Van Enennaam, A.L., De Figueiredo Silva, F., Trott, J.F., & Zilberman, D. (2020). Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay. Annual Review of Animal Biosciences, 9, 453-478. https://doi: 10.1146/annurev-animal-061220-023052.

Wang, K., Ouyang, H., Xie, Z., Yao, C., Guo, N., Li, M., Jiao, H., & Pang, D. (2015). Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Scientific Reports 5, 16623. https://doi: 10.1038/srep16623.

Wang, K., Tang, X., Liu, Y., Xie, Z., Zou, X., Li, M., Yuan, H., Ouyang, H., Jiao, H., & Pang, D. (2016). Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Molecular Therapy Nucleic Acids, 5, e396. https://doi: 10.1038/mtna. 2016.101

Wang, K., Tang, X., Xie, Z., Zou, X., Li, M., Yuan, H., Guo, N., Ouyang, H., Jiao, H., & Pang, D. (2017). CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Research, 26, 799–805. https://doi:10.1007/s11248-017-0044-z

Wang, H., Shen, L., Chen, J., Liu, X., Tan, T., Hu, Y., Bai, X., Li, Y., Tian, K., Li, N., & Hu, X. (2019). Deletion of CD163 Exon 7 Confers resistance to highly pathogenic porcine reproductive and respiratory viruses on pigs. International Journal of Biological Science, 15, 1993-2005. https://doi:10.7150/ijbs.34269. eCollection 2019.

Wang, Q., Vlasova, A.N., Kenney, S.P., & Saif, L.J. (2019). Emerging and re-emerging coronaviruses in pigs. Current Opinion in Virology, 34, 39-49. https://doi: 10.1016/j.coviro.2018.12.001.

Whitworth, K. M., Rowland, R. R. R., Ewen, C. L., Trible, B. R., Kerrigan, M. A., Cino-Ozuna, A. G., Samuel, M.S., Lightner, J.E., McLaren, D.G., Mileham, A.J., Wells, K.D., & Prather, R.S. (2016). Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature Biotechnology, 34, 20–22. https://doi: 10.1038/nbt.3434

Whitworth, K. M., Rowland, R. R., Petrovan, V., Sheahan, M., Cino-Ozuna, A. G., Fang, Y., Hesse, R., Mileham, A., Samuel, M.S., Wells, K.D., & Prather, R.S. (2019). Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic Research, 28, 21–32. https://doi: 10.1007/s11248-018- 0100-3

Xiang, G., Ren, J., Hai, T., Fu, R., Yu, D., Wang, J., Li W, Wang H, & Zhou Q. (2018). Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs. Cellular and Molecular Life Sciences, 75, 4619–4628. https://doi: 10.1007/s00018-018-2917-6

Xie, Z., Pang, D., Yuan, H., Jiao, H., Lu, C., Wang, K., Yang, Q., Li, M., Chen, X., Yu, T., Chen, X., Dai, Z., Peng, Y., Tang, X., Li, Z., Wang, T., Guo, H., Li, L., Tu, C., Lai, L., & Ouyang, H. (2018). Genetically modified pigs are protected from classical swine fever virus. Plos Pathogens, 14, e1007193. https://doi: 10.1371/journal.ppat.1007193

Xu, K., Zhou, Y., Mu, Y., Liu, Z., Hou, S., Xiong, Y., Fang, L., Ge, C., Wei, Y., Zhang, X., Xu, C., Che, J., Fan, Z., Xiang, G., Guo, J., Shang, H., Li, H., Xiao, S., Li, J., & Li, K. (2020). CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. Elife, 9, e57132. https://doi: 10.7554/eLife.57132

Yang, H., Zhang, J., Zhang, X., Shi, J., Pan, Y., Zhou, R., Li G, Li Z, Cai G, & Wu Z. (2018). CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus. Antiviral Research, 151, 63–70. https://doi: 10.1016/j.antiviral. 2018.01.004.

Yang, H., & Wu, Z. (2018). Genome Editing of Pigs for Agriculture and Biomedicine. Frontiers in Genetics 9, 360. https://doi:10.3389/fgene.2018.00360. eCollection 2018.

Yen, C.H., Tai, H.C., Peng, S.H., Yang, T.S., & Tu, C.F. (2020). Scaffold derived from GGTA1 and CMAH double knockout pigs elicits only slight inflammation in a gene-edited pig model. Materialia 14, 100836. https://doi.org/10.1016/j.mtla.2020.100836.

Yoon, C.H., Choi. H.J., & Kim, M.K. (2021). Corneal xenotransplantation: Where are we standing? Progress in Retinal and Eye Research 80, 100876. https://doi:10.1016/j.preteyeres.2020.100876

Zhao, J., Lai, L., Ji, W., & Zhou, Q. (2019). Genome editing in large animals: current status and future prospects. National Science Review, 6, 402–420. https://doi.org/10.1093/nsr/nwz013.

Zhang, Q., Lin, J., Huang, J., Zhang, H., Zhang, R., Zhang, X., Cao, C., Hambly, C., Qin, G., Yao, J., Song, R., Jia, Q., Wang, X., Li, Y., Zhang, N., Piao, Z., Ye, R., Speakman, J.R., Wang, H., Zhou, Q., Wang, Y., Jin, W., & Zhao, J.. (2017). Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proceeding National Academy Sciences USA, 114, E9474-E9482. https://doi.org/10.1073/pnas.1707853114.

Zhang, Q., & Yoo, D. (2015). PRRS virus receptors and their role for pathogenesis. Veterinary Microbiology, 177(3-4), 229-41. https://doi:10.1016/j.vetmic.2015.04.002.

Zhu, X., Wei, Y., Zhan, Q., Yan, A., Feng, J., Liu, L., Lu, S.S., & Tang, D.S. (2020). CRISPR/Cas9-mediated biallelic knockout of IRX3 reduces the production and survival of somatic cell-cloned Bama minipigs. Animals, 10, 501. https://doi: 10.3390/ani10030501

Zou, Y., Li, Z., Zou, Y., Hao, H., Li, N., & Li, Q. (2018). An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects. Biochemical and Biophysical Research Communication, 498, 940–945. https://doi:10.1016/j.bbrc.2018.03.085

Zou, Y.-L., Li, Z.-Y., Zou, Y.-J., Hao, H.-Y., Hu, J.-X., Li, N., & Li, Q.Y.. (2019). Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination. Journal of Integrative Agriculture, 18, 1329–1336. https://doi.org/10.1016/S2095-3119(19)62694-8