A natural mutation of the myostatin (MSTN) gene in some cattle breeds results in double muscling [39,40]. Czaja et al [41] recently revealed that MSTN may regulate pituitary development and function and that its inhibitory actions in muscle may be partly mediated by attenuating growth hormone action in the liver, leading to the expression of insulin-like growth factor 1 (IGF 1). In pigs, MSTN, IGF2 repressor Zinc finger BED-type containing 6 (ZBED6) and Fbox protein 40 (Fbox 40) gene knockout (KO) can improve growth or muscle mass production, but Iroquois homeobox 3 (IRX3) KO does not have this effect, as summarized in Table 1.

The first successful case of pig MSTN KO was achieved by using ZFN in the Meishan breed, and the resultant homozygotes reached adulthood normally but showed a higher percentage of lean body mass growth, exhibiting wider dorsal musculature and double muscling of the hip in particular [42]. However, the same attempt to achieve MSTN gene KO by using CRISPR/Cas9 failed to produce healthy KO piglets in the Landrace and Large White breeds [43,44]; but they later successfully generated 23 Erhualian ethnic breed pigs showing obvious muscular protrusion, a wider back and fuller hips relative to non-KO pigs [46]. Since Wang et al [45] suggested that commercial breeds would be more sensitive to the KO of endogenous genes, Zou et al [46] generated two healthy Duroc pigs with the Belgian Blue mutation only in heterozygosity, but they did not show double muscling at the neonatal stage. It could be explained by a recent finding that showed in commercial MSTN KO pigs, the decreased expression level of type I collagen and Scleraxis, could result in umbilical hernia and tippy-toe standing problems typified by the tendon and linea alba dysfunction [47].

Similarly, using CRISPR/Cas9, Xiang et al [48] mutated the IGF2 intron 3–3072 site of the IGF2 gene, which abolished ZBED6 repressor binding, causing the loss of its regulatory function, in indigenous Bama minipigs and obtained healthy animals with an improved growth rate, higher lean content, and little change in meat quality. This was the first report that editing noncoding regions could improve economic traits in pigs of indigenous breeds. Furthermore, Li et al [49] used CRISPR/Cas9 editing and introduced two mutations (PVD20H and GP19del) in the MSTN signal peptide region in Liang Guang Small Spotted pigs, an indigenous Chinese breed, without inhibiting mature MSTN production. This approach downregulated MSTN^+/PVD20H and MSTN^KO/PVD20H and upregulated myogenic regulatory factors, including MyoD, Myo-genin, and Myf-5. The precise editing of the MSTN signal peptide enhanced porcine muscle development without markedly affecting the expression of the mature MSTN peptide. This might be a better KO approach applied to commercial breeds for the further improvement of lean body mass without disturbing their normal physiology, such as causing reproductive disorders or increasing stress susceptibility, as observed in the highly muscled pig breed Pietrain.

Fbox 40 in mice targets insulin receptor substrate 1 (IRS1) for ubiquitylation and degradation, and the abnormal expression of FBOX 40 in humans is associated with muscle pathology, causing limb-girdle muscle dystrophy [50]. Zou et al [51] performed Neo resistance gene knock-in (KI) into exon 4 of FBOX 40 to abolish its gene function and identified increased IRS1 expression and a 4% increase in muscle mass growth. The authors recapitulated human muscular disease and suggested the application of this strategy in pig production. Similarly, the IRX3 gene is implicated in human obesity and controls body mass and body composition in mice, but Zhu et al [52] found that clones of IRX3^−/− Bama minipigs showed a significantly decreased birth weight, poor viability, and short survival after farrowing. The disparity in these results might originate from errors in somatic cell nuclear transfer (SCNT), although the authors hypothesized that IRX3 may be responsible for some important physiological functions in pigs and should not be targeted as a gene-editing candidate for body fat reduction [52].

Domestic pigs have no brown adipose tissue (BAT) and show no uncoupling protein 1 (UCP1) expression in their mitochondria [53,54], and exons 3 to 5 of the UCP1 gene were deleted during evolution 20 million years ago [55]. The function of UCP1 is to disengage oxidative phosphorylation from ATP synthesis in mitochondria and dissipate energy as heat in BAT for survival in cold environments or under cold stress. Although Lin et al [56] proved that cold adaption in pigs depends on UCP3 in beige adipocytes, pigs show poor thermoregulation [55] due to the absence of nonshivering thermogenesis; thus, high thermoneutrality is needed. The provision of additional warmth by various means to keep piglets warm is a basic practice of producers, especially in temperate regions. Zhang et al [57] constructed a porcine adiponectin promotor with mouse UCP1 cDNA and performed KI of the exogene at the porcine UCP1 exon 2 site through GE by using CRISPR/Cas9. The UCP1 KI piglets could maintain a normal rectal temperature of 38°C during 4 h of 4°C cold exposure, whereas the control group showed hypothermia (2°C lower). Since the adiponectin promoter drove UCP1 expression in adipocytes, the KI pigs grew normally relative to the control pigs, without noticeable harm to their well-being during the 6-month study period. Carcass evaluation further showed that backfat thickness, adipocyte size, and body fat accretion were all significantly decreased, resulting in an improvement in the carcass lean percentage. The authors also claimed that KI pigs showed a loss of body fat upon UCP1 activation in white adipocyte tissues, which would further improve pig welfare and reduce economic losses due to external energy expenditure to achieve thermoneutrality, especially in post farrowing piglets.

Red meat (pork, beef, and mutton) consumption may induce allergies due to the presence of alpha-Gal on the muscle cell surface [58]. This uncommon alpha-gal syndrome [59] can be avoided by choosing a product from which the antigen has been removed in KO livestock. Recently, the FDA approved the commercialization of GGTA1 gene-edited or KO pigs, also known as GalSafe pigs, for pork consumption [60]. This is the first case of a livestock biotechnology product launched for both food and biomedical uses.

The red meat cell surface expresses tremendous amounts of N-glycolylneuraminic acid (Neu5Gc; nonhuman glycan) [61], which, once absorbed, is incorporated on the surface of human cells [62] and elicits chronic inflammation, causing a major risk of colorectal cancer and atherosclerosis development [63]. Transforming Neu5Gc to Neu5Ac (the human form) in pigs through the KO of the responsible CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene has been a goal of xenotransplantation, and in animal production, this would transform red meat into white since poultry and fish also show the Neu5Ac form. Our research group has generated alpha-gal [64] and Neu5Gc [65] KO pigs by CRISPR/Cas9 GE. Through crossbreeding, dKO offspring without any major pathophysiological indications were obtained. After comparing the intestinal decellular scaffold (extracellular matrix, ECM) from dKO and wild-type (WT) pigs, we implanted dKO or WT ECM into dKO recipient longissimus and found that the dKO ECM evoked less inflammation than the WT ECM [66]. It is suggested that the dKO pigs will provide better medical grafts than WT pigs and that the dKO pigs can be considered an animal model for studies of alpha-Gal allergy and may also recapitulate human sialic biology to a greater extent in both healthy and diseased conditions [67]. Additionally, these dKO pigs could be served as a heathy red meat source.

Recent efforts of exogenes KI at Rosa26 locus by CRISPR/Cas9, healthy and tasty pork could also be resulted [68,69]. You et al [68] generated dual, Fat1 and IGF-1, transgenic (TG) pigs that could provide pork with a significantly higher ω-3 polyunsaturated fatty acid (PUFA) level and a significantly lower ratio of ω-6 PUFA/ω-3 PUFA. Gu et al [69] generated TG pigs carrying muscle-specific overexpression of peroxisome proliferator-activated receptor gamma2 (PPARγ2), which significantly increased intramuscular fat content while maintaining carcass lean ratio. Although, KI the PPARγ2 gene is considered TG in pigs, yet, both sequences of promoter and PPARγ2 gene are based on pig genome [68] and thus the transgenes are “safe-harbored” in Rosa26 locus, making the pork safe as the ordinary.

PRRSV emerged in the late 1980s and rapidly became an epidemic devastating the pig industry globally. In vivo, the virus shows very narrow cell tropism, targeting specific subsets of porcine monocytes/macrophages, and it infects the cells via the heparan sulfate, sialoadhesin (CD169) and CD163 receptors [81]. The CD169 KO pigs generated via traditional homologous recombination and SCNT were not resistant to PRRSV infection, suggesting that CD169 is not necessary for PRRSV infection [82]. To date, CD163 on porcine macrophages has been the best-studied receptor involved in PRRSV infection [83]. Efforts including the KO of CD163 [70,71,75,76], deletion of exon 7 (scavenger receptor cysteine-rich domain 5 region of the CD163 protein) of the CD163 gene [71,74], and the deletion of a portion of exon 7 in the infective pocket of virons [73] have achieved full resistance to PRRSV infection without disturbing the well-being of GE pigs [72–74,84–86]. Furthermore, Xu et al [75] generated dKOs of CD163 and pAPN (porcine aminopeptidase N, a factor responsible for TGEV infection) and proved that dKOs pigs could be resistant to type II PRRSV and TGEV infection.

When infected by ASFV, domestic pigs and Eurasian wild boars (Sus scrofa) develop a lethal hemorrhagic fever, whereas this is not observed in warthogs (Phacochoerus africanus) or bush pigs (Potamochoerus larvatus), which do not develop marked clinical signs; the last two species evolved in southeastern Africa in a sylvatic cycle with a vector of ASFV the common and argasid ticks of the Ornithodoros moubata species complex that live in their burrows and in the pens of domesticated pigs. ASFV has gradually became widespread in eastern and central Europe while showing much faster outbreaks in China and other countries in Asia [8]. Macrophages have been identified as the target cells of ASFV [87], and antibodies against CD163 are able to inhibit both ASFV infection and viral particle binding to alveolar macrophages, highlighting the role of this molecule as a putative receptor for the virus [88]. However, Popescu et al [78] challenged CD163 KO pigs with the Georgia 2007/1 ASFV isolate and failed to reveal any resistance to viral infection, and the observed clinical signs, mortality, pathology, and viremia differed little between KO and WT pigs. A group at the University of Edinburgh has identified polymorphic variation in RELA (p65; v-rel reticuloendotheliosis viral oncogene homolog A), the major component of the NF-κB transcription factor and revealed that three AAs of RELA differ between warthogs and domestic pigs [89]. Subsequently, they generated live RELA KO pigs by TALEN and ZFN editing [90] and substituted three AAs of domestic pig RELA with warthog AAs by using CRISPR/Cas9 [77]. However, the same research team recently proved that the 3 AAs substitution was not sufficient to confer resilience to ASFV; it only delayed the onset of clinical symptoms and resulted in less virus in nasal secretions and blood in some animals [91]. On the other hand, a German group transfected wild boar lung cell lines with a plasmid encoding Cas9 and a guide RNA targeting codons 71 to 78 of the phosphoprotein p30 gene (CP204L) of ASFV and demonstrated that ASFV plaque formation was completely abrogated, and virus yields were reduced by four orders of magnitude due to targeted Cas9 cleavage of the virus genome [92]. In such an approach, Cas9 and the guide RNA plasmids need to be integrated into the cell genome to maintain long-term resistance to ASFV infection. Thus, once the cells are cloned into animals, they might be argued to be GMOs with a high probability of off-target effects due to the integration and expression of the Cas9 gene. Recently, Xie et al [80] used a CRISPR/Cas9-mediated KI strategy to generate TG pigs carrying antiviral small hairpin RNAs (shRNAs), safely integrated at the porcine Rosa26 (pRosa26) locus, to test the resistance to anti-CSFV. They found that in the TG pigs subjected to in vitro or in vivo CSFV challenge, the replication of CSFV was effectively limited, as demonstrated by reduced CSFV-associated clinical symptoms and mortality. Furthermore, this disease resistance could be stably transmitted to the F1 generation. A similar approach should effectively generate ASFV-resistant pigs, but the outcome remains to be seen.

Four genera of coronaviruses (CoVs), including α-, β-, γ-, and δ-coronaviruses, have been identified. Currently, six CoVs are known to infect pigs, including four α-coronaviruses (TGEV, porcine respiratory coronavirus [PRCV], PEDV, and swine acute diarrhea syndrome-coronavirus [SADS-CoV]), one β-coronavirus (porcine hemagglutinating encephalomyelitis virus [PHEV]), and one porcine δ-coronavirus (PDCoV), which cause different types of infections of great commercial concern in pigs [93]. Among these viruses, TGEV, PRCV, and PHEV have globally circulated in pig populations for decades with few clinical effects, whereas PEDV, PDCoV, and SADS-CoV are considered emerging CoVs and cause severe acute diarrhea in piglets with high mortality. These viruses infect host target cells via S-protein binding to the proposed receptors of aminopeptidase N (APN) and Neu5Gc; however, porcine GE by CRISPR/Cas9 for APN [76,79] or CMAH [65] KO did not achieve PEDV infection resistance, indicating that these receptors might not be sufficient for PEDV infection. However, APN KO pigs could resist TGEV infection [75,79] and showed decreased susceptibility to PDCoV infection with normal growth performance [75]. CoVs are RNA⁺ viruses, and their S-protein is a glycoprotein that undergoes complicated posttranslational modifications to achieve diverse antigenicity [94]. These highly variable sequences [95] hinder effective vaccine development.

The challenges of precisely editing the genome, developing effective vaccines, and designing a strategy ensuring biosecurity in the face of ASFV and CoVs threats remain in pig production. As mentioned above, Xie et al [80] performed the CRISPR/Cas9-mediated knock-in of anti-CSFV antiviral shRNAs and achieved effective CSFV infection resistance. Using this TG strategy, Hu et al [96] generated TG pigs constitutively expressing FMDV-specific shRNAs and found that these animals showed higher resistance to FMDV. It appears that combining shRNA and GE to target and degenerate the critical region of the virus genome, without the integration of the Cas9 gene, could be a viable strategy for achieving resistance in animals to lessen or even prevent infection.