The Application and Trend of Pig Science Trough Precise New Breeding Technology

The Application and Trend of Pig Science Trough Precise New Breeding Technology

Published: 2021.09.13
Accepted: 2021.09.13
Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan
Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan


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.


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 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."


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.


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.


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.  


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