Genetic engineering techniques have been used in research to increase the understanding of genetic function, diseases, and productivity. Early attempts of genetic engineering were mainly in laboratory animals such as mice, where genetic engineering was carried out by using exogenous recombinant vector form or packing vector that would be introduced into viruses to infect cells or in vitro fertilized embryos (1). However, at that time, the exogenous genes were randomly inserted into the genome, an approach that had several limitations including low efficiency, unwanted genome insertion, and sometimes, germline transmission did not occur or was silenced over the generations. In addition, the early technology required a lot of time and resources, and sophisticated gene engineering could not be carried out. Consequently, it was difficult to apply gene engineering technology to livestock. The discovery of gene scissors revolutionized gene engineering technology and overcame limitations of the traditional technology. Zinc Finger Nucleases (ZFNs), the first gene editing tool, was applied to rodents in 2009 (Rat) and 2010 (Mouse) (2, 3), and showed great potential. ZFNs have several shortcomings, such as low efficiency and cytotoxicity; however, almost all of ZFNs’ limitations were solved with the emergence of Transcription Activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. Impressively, CRISPR/Cas9, which was discovered in 2012, can be applied in eukaryotic cells (4, 5). Overall, gene editing technology made gene editing and production of genetically engineered livestock easy and quick (6).

Genetically engineered livestock were produced using gene editing technology, mainly somatic cell nuclear transfer (SCNT) or microinjection (MI). SCNT has the advantage of being able to select somatic cells with a desired gene mutation at the somatic cell level in advance; however, it is limited by frequent absorption, death before and after delivery, and the production of abnormal offspring. On the other hand, with MI, it was possible to produce a healthy individual, but it takes a relatively long time to obtain a complete mutation because of mosaicism. Therefore, the choice of method for experimental research may vary depending on the purpose of the experiment.

Gene engineering technologies have rapidly evolved and gained momentum in livestock improvement. These advancements have led to the creation of genetically engineered livestock animals with enhanced traits, disease resistance, and improved productivity (7-10). This review article aims to provide an overall understanding of the past and present genetic engineering technologies in livestock and up-to-date research findings. Furthermore, we aim to convey the content of the regulatory framework for livestock applied in these technologies, in particular the recent FDA approval.

Viruses and homologous recombination

Gene engineering technology is a method of overexpressing or inhibiting a specific gene and has been used for research to increase understanding of gene functions. In 1972, cloning of recombinant genes was established (Fig. 1A), and in studies conducted two years later, transgenic mice induced by overexpressing the exogenous gene (simian virus [SV] 40 DNA) (Fig. 1A). In this study, SV40 DNA was identified in mouse tissues, but the germline transmission of the mouse was not identified (11). In 1976, the same research group injected the Moloney Leukemia virus into a fertilized embryo, which resulted in the successful introduction of mice with the virus vector. Furthermore, the study reported the presence of the introduced genes in the offspring of these mice. This groundbreaking study demonstrated, for the first time, the possibility of exogenous gene insertion and germline transmission (12).

However, gene modification using viruses had drawbacks such as experimental safety concerns, unwanted integration into the host genome, and potential for oncogenic effects (13, 14). To improve gene engineering technologies, homologous recombination, where nucleotide sequences are exchanged between two similar or identical DNA molecules, has been applied for more precise gene insertion or deletion in rodents and livestock (Fig. 1A). Despite these ongoing improvements, continuous enhancement is required to achieve efficient and precise insertion of the desired genes into a specific host genome. To produce genetically engineered livestock through SCNT or microinjection (MI) in a homologous recombination method, exogenous genes must be introduced into somatic cells or IVF embryos. Homologous recombination occurs as cells divide, and a series of screening processes are needed over an extended period for gene engineered cell or embryo selection due to the probability of low efficiency (15). However, long-term selection easily causes cell aging and death in somatic cell and embryos (16, 17). Consequently, it was difficult to produce genetically engineered livestock animals with high efficiency in 1990s (18-20).

The advent of engineered nucleases

A study published in 2009, showcased the application of ZFNs as the first-generation gene editing tool to suppress specific genes in experiment animals (Fig. 1A) (2, 3). ZFNs showed promising results as a tool for targeted gene manipulation, opening new possibilities for precise genetic engineering in animals. Representative examples are studies on the production of new varieties or methods of treating mutations by producing animals with mutations (21). While the existing gene engineering methods carry the risk of unexpected random insertion by introducing exogenous recombinant plasmid (Fig. 1B) (22), when gene scissors were introduced, it was possible to produce gene knockout livestock without unwanted introduction of exogenous genes (Fig. 1C). Furthermore, gene editing efficiency is higher than conventional homologous recombination and speeds up the process of long-term somatic cell culture for screening gene engineered cells (23). Therefore, the group that studied animal production with the desired trait through SCNT focused on animal production by continuously applying gene editing tools to SCNT because it can reduce the aging of somatic cells compared to before. On the other hand, a research group that used in vitro fertilization (IVF) to manipulate genes conducted a study that efficiently applied ZFNs in IVF embryos (24).

After the advent of ZFNs, the next generation of engineered nucleases like TALENs and CRISPR/Cas9 emerged (25). Like in TALENs, a second-generation gene editing tool, gene editing is performed in a manner similar to ZFNs, but experimental design is relatively simple, which means it takes less amounts of time and is laborsaving. CRISPR/Cas9 is a third-generation gene editing tool, unlike ZNFs or TALENs, which require designing new engineered nuclease components every time depending on the target site, it is easy to change the target site through a simple design of a guide RNA (Fig. 1A) (25, 26). Therefore, among the three genetic scissors, it offers the greatest time-saving and laborsaving advantages. In addition, it has high specificity and efficiency by showing fewer off-target advent than ZFNs and TALENs (25-28).

Cell-mediated animal production: SCNT

The representative method for producing genetically engineered livestock is SCNT, which involves exchanging the nucleus of a somatic cell with the nucleus of an oocyte (Fig. 2A). In 1997, the first cloned sheep, Dolly, was successfully cloned using SCNT in a mammalian sheep (29). SCNT can be used not only for producing cloned livestock but also for creating genetically engineered animals using genetically engineered somatic cells. Furthermore, the efficiency of gene engineering was higher in somatic cells than when applied directly to IVF embryos. In the early 2000s, laboratory results of studies on genetically engineered somatic cells suggested that livestock could be created. In 2002, Alpha-1,3-galactosyltransferase (GGTA1) gene KO pigs were successfully produced through SCNT (30, 31), and many kinds of genetically engineered livestock animals were produced by SCNT after the advent of gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) (32-37).

While manipulating the desired gene in somatic cells is easier than in embryos, SCNT has associated side effects. These include abnormal reprogramming during embryogenesis, resulting in a low pregnancy rate and instances of sudden death after delivery (38, 39). To enhance the efficiency of SCNT, numerous attempts have been made, including addressing epigenetic factors to overcome reprogramming challenges. Among them, two studies focused on the injection of epigenetic factors during embryo development (40, 41). Nevertheless, the aforementioned issues have not been fully resolved to date, and SCNT still has a lower implantation rate than IVF (42).

Embryo-mediated animal production: MI or electroporation

Before the introduction of SCNT in livestock in 1997, genetically engineered animals were primarily produced through the MI of DNA into in vitro fertilized embryos (Fig. 2B). Several studies have demonstrated random insertion through MI in rabbits, pigs, and sheep (43), but achieving germline transmission to the next generation has not been successful (44, 45). Furthermore, the efficiency of MI was notably low in terms of high-throughput techniques by consuming more time. However, in some cases, MI is more efficient than electroporation in terms of mutation efficiency (46). To enhance efficiency of MI, lentivirus or retrovirus was injected into a zygote, demonstrating higher effectiveness compared to DNA injection.

Since the emergence of gene editing tools such as ZFNs, TALENs, and CRISPR/Cas9, gene engineering has become significantly more efficient and accurate. Moreover, the efficiency of producing genetically engineered livestock animals using MI has been greatly enhanced (47-49). With the advent of the ribonucleoprotein (RNP) complex within the CRISPR/Cas9 system, the application of one of the most common techniques for RNP delivery, electroporation, has become feasible (Fig. 2B) (9, 50, 51). The electroporation has enabled the production of gene-engineered livestock animals through a simpler method as opposed to the previously laborious MI (9, 52). Therefore, various types of gene-engineered livestock animals have been created using gene editing tools, resulting in enhanced productivity, disease resistance, and advancements in biomedicine. The next paragraphs provide illustrative examples from each respective field.

Productiveness

According to the World Agricultural Organization, the demand for animal proteins (meat, eggs, milk, etc.) is expected to increase to 1,800 million tons as the population increases. To meet these protein requirements, research and development efforts are needed to improve livestock productivity using genome editing tools (9, 53).

Traditional breeding methods have achieved significant improvements in livestock over time. However, recent active research has led to the discovery of genes related to productivity, and the engineering of such specific genes presents a novel direction to enhance productivity in livestock. The following are some of the studies that have been conducted to increase productivity according to species in livestock.

Cattle: In a study aimed at producing high-quality meat, the myostatin (MSTN) gene, which inhibits muscle growth, was edited in cattle using TALENs and SCNT, resulting in the successful birth of cattle with increased muscle mass in 2015 (54). Additionally, in 2022, CRISPR/Cas9 was used to induce MSTN mutations in in vitro fertilized embryos, leading to the successful production of beef cattle with increased muscle mass (55). From these gene editing results in cattle, it has been demonstrated that the MSTN mutation is transmitted to the next generation through the germline, laying the foundation for the mass production of MSTN mutation cattle (52). On the other hand, for only males to be born, an in vitro fertilized bovine embryo, in which the SRY gene was inserted into chromosome 17 using CRISPR/Cas9, was produced, and a report was published in 2021 that a calf was successfully born (56).

Holstein, a dairy breed of cow, absorbs a lot of light and is prone to high-heat stress. Heat stress has a significant negative impact on animal reproduction and growth, and as a result, numerous studies on solutions for such challenges are being conducted (57, 58). Accordingly, a mutation was induced in the PMEL gene, which determines black color in hair, using CRISPR-Cas9 and resulting in gray hair. In 2022, New Zealand researchers announced that calves with the PMEL mutation were successfully born, showing the actual color changes in their hair. In another method to mitigate heat stress, the SLICK gene, which is related to the length of hair, was edited in cattle (7, 59). Recently, the US FDA announced that it will soon approve cows produced after inducing SLICK gene mutations using gene editing, as they believe that the risk of any adverse effects from gene modification is very low (60, 61).

In 2011, ZFNs and SCNT were used to produce a cow that lacked beta-lactoglobulin (BLG), which is known as a major allergen in milk. Subsequently, the research was extended to produce actual milk from BLG knockout cows. In 2018, it was confirmed that the milk produced by these cows contained no beta-lactoglobulin (62, 63). Recently, as part of efforts to maximize productivity, our group successfully produced dairy claves with simultaneous mutations in both MSTN and BLG genes (9).

Pigs: Cloned pigs with MSTN gene mutation were successfully produced using ZFNs in 2015 (64), TALENs in 2016 (65), and CRISPR/Cas9 in 2017 (66). In addition to having relatively high muscle mass, all pigs with MSTN mutation also showed improved productivity. So far, there are many studies on the MSTN mutated pigs to increase muscle mass, especially in China (67-69).

Mutation of the Nanos2 gene, which is known to promote male cell differentiation and inhibit feminization, results in the birth of an animal that does not produce sperms in males. Based on this, Nanos2 was knocked out in mice, pigs, goats, and cattle, and it was confirmed that sperms were not produced in these animals (70). Furthermore, in 2020, a study was published in the US where spermatogonia stem cells derived from wild-type pigs were transplanted into pigs with a Nanos2 mutation that prevented sperms production. The study showed that the transplanted spermatogonia stem cells could differentiate and successfully produce sperms. Based on this study, the researchers can predict that a system will be prepared to mass-produce semi-permanently excellent sperms by separating spermatogonia stem cells derived from excellent breeds of males.

Sheep and goat: Goats are famous for providing meat and fur. In 2016, a goat with a MSTN mutation was produced using SCNT and TALENs and was reported to have increased production (34). Additionally, in 2015, in Uruguay, a study reported successful use of MI to produce a sheep with increased muscle mass (48). Moreover, in China, in 2016, a study reported successful multiple gene editing using MI in sheep (71). In another study, in a cashmere goat, double knockout for the MSTN gene that controls the amount of meat and the FGF5 gene that affects the length of hair was performed in the somatic cell stage and a transgenic goat was produced using SCNT (72).

Disease resistance

Recently, there have been numerous new pandemic diseases affecting humans. Similarly, animals have been affected by infectious diseases, such as foot-and-mouth disease. Consequently, there are active studies on improving disease resistance for diseases such as African Swine Fever Virus (ASFV), Pig Reproductive and Respiratory Syndrome Viruses (PRRS), Bovine Spongiform Encephalopathy (BSE), and tuberculosis. If a disease resistance model is established through gene engineering in livestock that are reared in a limited space and are vulnerable to the spread of disease, it can reduce time and economic loss by reducing dependence on antibiotics. It can also minimize the pain caused by disease and therefore improve animal welfare.

Cattle: In 2005, SCNT was used to produce a cow that was resistant to mastitis (breast inflammation), allowing the secretion of anti-inflammatory substances from the mammary glands. When these cows were exposed to mastitis, they were confirmed to be resistant (35). Based on the results of these studies, in China, in 2014, after gene-engineered somatic cells were used to secrete human lysozyme at the location of the casein gene that secretes milk using ZFNs, SCNT was processed using these cells and used to produce cows resistant to breast inflammation (73). In addition, a study related to the production of cows with prion resistance using bovine somatic cells that had mutated prion genes was published in 2007 (37).

Tuberculosis, also known as consumption, is one of the diseases that affect cattle and is causing a lot of damage to the livestock industry. To make cattle resistant to the disease, the resistant gene SP110 was inserted into a specific location on chromosome 28 using TALENs in bovine somatic cells. SCNT embryos were transplanted into 147 surrogate mothers, and 23 calves were produced. In 2015, a Chinese research group reported that the calves were resistant to tuberculosis because of actual infection tests on this newborn calf (74).

Isoleucyl-tRNA synthetase syndrome (IARS), a recessive genetic disease that affects Japanese black cattle, was corrected using CRISPR/Cas9 in bovine somatic cells, and cloned embryos using corrected somatic cells as donors were transferred (75). In 2017, After genetic testing in the fetus done by inducing miscarriage 34 days after transplantation, the IARS mutation was detected. Recently, the first gene-edited calf with resistance to a major viral pathogen, Bovine Viral Diarrhea Virus (BVDV), was successfully produced. BVDV is one of the significant viruses affecting the health and growth of cattle worldwide, ultimately leading to substantial economic losses (76).

Pigs: There are respiratory infectious diseases caused by various viruses, such as PRRS, pig circovirus, and pig influenza virus. Among these, PRRS causes significant economic losses to the pig industry. The CD163 is known to be a cellular receptor capable of mediating infection of the PRRS virus. In 2015, in the US, a pig with CD163 mutation was produced using SCNT and was confirmed to be resistant to PRRS (77). Later, similar results were announced in the UK in 2018 (78), and in China in 2019 (79), where pigs resistant to PRRS were successfully produced.

Biomedicine

Xenotransplantation: The most popular part of the biomedicine field is the production of xenotransplantation animals through gene engineering. Since pig production through SCNT is now possible, organ transplant research using pigs is an active research topic. In 2002, research on pig production using xenotransplantation by knockout the GGTA1 gene, which is known to cause acute immune rejection and act as an allergen in meat, was conducted. The GGTA1 knockout pig was produced using SCNT and homologous recombination. In addition, the US FDA’s announcement in December 2020 that GGTA1-suppressed pork produced from gene engineering pigs can be allowed as food in people with the allergens. This could be a starting point for using pigs produced through gene engineering as meat in the future. With the advent of gene editing tools, it has become relatively easy to produce specific gene-edited animals. In 2015, a research group in the US used CRISPR/Cas9 to completely remove porcine endogenous retrovirus (PERV) and successfully produced cloned pigs. In January 2022, a pig heart with various controlled genes became the world’s first organ xenotransplantation in a human (80). However, two months after the transplant, the patient died, warranting further research.

Duchenne Muscular Dystrophy (DMD) disease: Gene editing in livestock can be used as a human genetic disease model. For example, the DMD disease pig models can be used as a means of research for the cause of DMD, which mainly occurs in young people or to develop treatments. In 2021, a research group in China edited DMD exon 51 to create human-like DMD genetic diseases using CRISPR/Cas9. They successfully produced mutated piglets and disease phenotype in these pigs were very similar to that of human diseases (81).

Cardiovascular disease: Among the studies that have been conducted along with heterogeneous organ studies is the study of cardiovascular diseases using pigs. The PCSK9 gene, known to control LDL cholesterol levels, was overexpressed by SCNT in pigs, to produce and study a human cardiovascular disease model (82). In addition, in 2018, Chinese researchers announced that the APOE gene was suppressed using CRISPR/Cas9 and that it produced a miniature pig of an arteriosclerosis model (83). Similarly, in 2017, in China, SCNT-derived genetically engineered pigs with somatic cells that knocked out both APOE and LDLR genes were produced. Notably, serum patterns similar to human metabolic diseases were observed in these pigs (84).

Neurological disease: Recently, there have been several studies for neurological diseases through gene editing using pigs. This is because pigs are larger than rodents’ brains and have a similar structure and size to humans, making them suitable for research on neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases (85, 86). There was a case of gene overexpressing of hAPP, hTAU, and hPS1 genes in pig somatic cells in 2017 (87), and a study that produced PARK2 gene editing pigs using TALENs in 2014 (88), or simultaneously dropped Parkin, DJ-1, and PINK1 genes in 2016 for neurological diseases research (89).

The US FDA uses the term “intentional gene alternations (IGAs)” to describe genetic modifications in animals. IGAs in animal encompasses a modification of genomic DNA using modern molecular technologies which random or targeted DNA sequencing changes (90). Most of the IGAs in livestock are applied to human health, improved animal health and well-being, and enhanced production and food quality.

To date, the US FDA has approved five cases of IGAs in animals (Table 1), with two approved for use as food and three approved as pharmaceuticals (91). To receive the FDA approval, rigorous regulation must be followed. The approval process for the products took more than a decade to complete and was costly (92). However, currently, IGAs animal products approvals are granted frequently in each county, while the FDA still closely monitors the process (93). The FDA seeks to find a risk-based approach and focuses on the balance between regulatory responsibilities and the need to get innovative products to the market efficiently. Recently, the FDA officially announced the low-risk determination for marketing of genome-edited beef cattle products. The cattle with edited PRLR gene have short hair to resist heat stress (61, 94). In addition, the FDA has given Washington State University (WSU) researchers the first-of-its-kind approval to feed five gene-edited pigs to people (95). There is a clear distinction between the previous FDA approvals for IGAs in animals and the current cases. In the previous approvals, exogenous genes were inserted into the animals, while in recent cases, genetic editing is performed on the animals’ existing genes. The ongoing approvals of IGAs in animals are expected to contribute to building public trust and serve as innovative steps in the development of new foods.

In early genetically engineered livestock production research, various genetic engineering technologies such as vector random insertion, virus infection, SCNT, and MI were applied, but there were frequent cases of low efficiency and production of abnormal individuals. The development of molecular biology saw the emergence of gene editing tools which have improved the low efficiency of the previously applied gene engineering technologies and significantly reduced the time and labor burden. Consequently, the production of genetically engineered livestock that were difficult to try in the past has been achieved without health concerns.

However, in livestock, developed molecular biology and more efficient gene engineering technologies are still required to enable stable establishment of embryonic stem cells, improved SCNT efficiency, and more efficient and safer gene editing tools. In addition, for such a theoretical and technical leap forward, in-depth consideration of global regulations and approval for the precise stability of genetically engineered livestock and its application in human society will be needed to accelerate the production and market of genetically engineered livestock.

This study was financially supported by the National Research Foundation of Korea (NRF-2021R1F1A1051953) and the Materials/Parts Technology Development Program (20023353, Development of composite formulation with a sustained release [gene] for the treatment of companion animal sarcopenia) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea). All of the figures in this paper were created with BioRender.com.

The authors have no conflicting interests.