Abstract

Food security continues to remain a critical challenge confronting humanity. Worldwide, the demand for food products is extensively increasing. Today, the research of generating transgenic organisms is one of the fastest-developing biotechnology fields, as it promises sustainability of agriculture and aquaculture. Many of the animals observed today, including top-quality agricultural breeds such as Holstein cows and Landrace swine, have undergone significant changes and are entirely distinct from their wild ancestors. The reason behind this is that they went through decades of selective breeding (SB) techniques, particularly crossbreeding and mutation breeding. Genome or gene editing (GE) refers to the process of targeted restructuring of a gene at a specific site through genetic engineering (GEN) technology. It ultimately leads to the construction of ‘custom-made animals’ with better hereditary features and amendments. It effectively imitates the natural methods that form the origin of SB programs. However, farmers’ attempts to provide stable productivity and sustainability using traditional breeding proved difficult, inefficient, and time-consuming. So, there is a pressing need for novel strategies for efficient animal breeding today. Compared to conventional breeding techniques, GE is receiving considerable attention, since it can efficiently adjust the target gene to get a GEN animal with desired traits. Developments in animal breeding methods are ongoing, and as each innovation is made, it potentially offers a range of opportunities. The prospects vary between providing targeted production traits such as yield and nutritional profile, improving disease resistance, and expanding animal welfare. This article gives an overview of GE techniques used in breeding livestock models, birds, and fishes. It concludes that applications of GE to improve the traits are the present and future of innovation in animal production. The limitations and future of the breeding process and the development of GE technology in farm animal breeding are also addressed.

1. Introduction

The United Nations predicted that the world population will grow to 9.7 billion by 2050 and reach 11.2 billion in 2100 [1]. According to the Food and Agriculture Organization (FAO) of the United Nations [2], the need for more or better animal proteins such as (eggs, meat, milk, etc.) is expected to rise to 1,800 million tons while the population increases [3]. Significant threats to animal production practices are added by the destructive impacts of climate change, increased urbanization, and political conflicts [4,5]. The cumulative effect of these developments in agriculture and food production is anticipated to be between 800 billion and 1.2 trillion over the next 20 years, accounting for about 36 percent of the total potential impact [6,7]. Given all this, the global demand for food security will substantially grow as well. One potential solution to this problem is the development of sustainable aquaculture and agricultural resources. In addition, there is an increasing focus on creating substitute, such as lab-grown meat, to relieve environmental pressures from traditional livestock and seafood production [7].

Aquaculture produced from fisheries for human use is the quickest-growing food-making sector and promptly becomes a principal element for international seafood security because of the elevated animal protein in human nutrition [2,8]. In contrast to land-based agriculture, which competes with habitable land areas, aquaculture has the advantage of having enormous, accessible ocean areas. Another benefit of fish farms is that they have a higher adequate feed conversion level than other food production methods [2].

Traditionally, scientists have used two significant strategies i.e., directed selection breeding and crossbreeding, to develop key traits of fish, poultry, and livestock [9]. However, these approaches could not provide an effective solution, mainly due to the inherent drawbacks, such as slow genetic progress, animals’ long cycle times, and prediction complexity [10] (Table 1). The classical methods have been incapable of introducing or improving high-quality genes rapidly and precisely without introducing undesirable genes. Some bad genes include those that would result in trait deficiencies in animals, such as low growth, infertility, susceptibility, etc.) [11]. For example, selective breeding for over 60 years has produced high-quality meat. However, it has also caused the buildup of recessive mutations that brought about genetic diseases [12]. Moreover, traditional strategies were inadequate for breeding certain traits, such as disease resistance. Resistance to numerous diseases is a complex trait controlled by more than one gene, making conventional genetic selection for disease-resistant breeding much more expensive, time-consuming, and inefficient [13].

Table 1:

A brief comparison between the significant available conventional and existing GE strategies used in animal breeding.

Aspect	Conventional Breeding Strategies	Gene editing Existing Strategies
Genetic change	Selects desirable traits that have occurred by natural mutation	Deliberate development of characteristics that are difficult to achieve by traditional breeding methods.
Major strategies in breeding	Directed selection breeding and crossbreeding	Gene editing: Knock-in/ knock out particular gene(s)
Insertion of the desired gene in the genome	Random at multiple sites	Site-specific and can target single and multiple sites
Genetic consequences	The process is slow and may result in the loss of many other existing desirable traits.	Fast and specific for removing undesirable genes
Off-target modifications	high	Less
Risk of introducing bad genetic traits like mosaicism and disease resistance	High	Less
Long-term risk of random insertion of undesired exogenous genes	High	Less risky, but not fully known
Possible threats to animal welfare	unlikely	higher
Efficiency at modifying the DNA of cells	Low and Poor	Much Higher
Time required to choose and stabilize genetic traits in a population	Long: Slow and requires more than one generation	Short: Fast and requires only one generation
Incidence of stillborn female	High	Not existing
Cost of breeding	Expensive	Less cost
Safety concerns about transgenic foods	Safe	Questions remain

To get out of the bottleneck and the associated technical problems, traditional breeding methods such as artificial selection and multi-generation hybridization have been replaced by more precise genome editing/gene editing (GE) breeding techniques. Genetic engineering covers a broad range of techniques that enable targeted modifications in an organism’s DNA (gene recombination). Using GE approaches, targeted mutations can be achieved in particular genes, and new lines of animals with important phenotypes can be produced. Gene editing technology has been deployed to create more innovative solutions for expanding the genetic improvement of fish and livestock, thereby promoting disease resistance in bred animals.

Thanks to gene editing methods such as zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR), significant advancements have been made in genetic modification. Later, other GE methodologies, such as base editor (BE) and prime editor (PE), were introduced into the field, but they are not widely used and are being tested and evaluated.

Research continues on the GE tools to enhance the effectiveness of targeted animal modifications, increasing the number of available GEN animal models. Figure 1 presents some of the milestones in animal breeding. The recent development of GE, along with the animal production technologies, gives the potential for speeding up successful animal genetic development. This development includes modification of production traits, disease resistance breeding, and improvement of animal health [14]. Despite the many profits of GE breeding, several concerns remain regarding the bioethics and biosafety of transgenic foods that carry foreign genes [15,16].

Embryonic stem cells (ESCs) can be grown in vitro for many passages and transformed with transgene constructs, causing genome modifications. The constructs used allow the selection of effectively transformed cells and permit gene targeting to be achieved. Consequently, genes can be precisely introduced, replaced, or deleted (so-called knock-ins (KIs) and knock-outs (KOs), respectively [17]. The injection of genetically modified embryonic stem cells (ESCs) into blastocysts, primarily through gene targeting, results in various genetic modifications. Many laboratories globally have tried to construct ESC from farm animals for several years. However, no robust and reproducible procedure has been published in this regard. Even in mice, the assembly of ESC is an expensive and labor-intensive technology [18]. Among farmed animals, the ability to engineer the genome has been restricted in livestock species. This restriction is due, in part, to the shortage of stable ESC that can give the germline [19]. As the actual embryonic stem cells (ESCs) capable of donating the germline have not been fully characterized in livestock species, their use remains limited. The expertise in targeted genetic modification of somatic cell nuclear transfer (SCNT) [20] permitted the generation of genetically engineered livestock. However, although this was possible, the efficacy of producing GEN animals using this route was poor; thus, only selective GEN models have been constructed [21].

Genetic engineering in agriculture refers to actively producing valuable plants or animals. Agricultural production implies cultivating crops and breeding animals for food or other resources, which are then distributed to markets for public use. Like plants and animal modification, attempts to breed plants and animals started hundreds of years before people recognized what DNA was. Genome editing technologies offer scientists a rapid and precise method to modify definite features of plants and animals, letting agriculture remain sustainable and productive in the face of a fluctuating climate and increasing world population pressure [25]. These technologies significantly impact agricultural farming since they can breed plant and animal lines with valuable traits that will display new settings, boost yields, generate higher-quality food, and deliver items for new markets. The applications of GE strategies, particularly TALENs, in several plant species, have been discussed in a previous chapter [26]. A brief comparison of genome editing (GE) approaches in plants and animals reveals that the methodology for genome editing in farm animals differs significantly from that in plants [27]. This difference is partly due to biological variations between the two kingdoms, like the mode of reproduction (for instance, plants can self-pollinate) and the number of offspring per reproduction cycle. Regarding GE traits, one transgenic plant transformation can re-develop from somatic cells, while animals can only grow from germline cells. Another significant difference between plant and animal breeding is that plant breeders focus on developing a distinguishable plant variety, often secured by plant breeders’ rights. In contrast, animal breeders focus choice on elite animals in the breeding scheme, with millions of genetically discrete individuals produced primarily by the multiplication of elite genetics via outcrossing [23].

The applications of GE technologies for livestock production offer huge promises. They are very diverse, varying between providing targeted production traits such as yield and nutritional profile, improving disease resistance, and expanding animal welfare. However, it is difficult to construct specific gene-targeted livestock because of technical difficulties and the long generation time of large animals. It also raises serious safety concerns related to the expansion and use of genetically modified products in contrast to conventional breeding, which is envisioned as safe [28,29].

There is an ongoing debate on the potential of GE technologies and their association with other methods in agriculture for developing food security. A Genetically Modified Organism (GMO) is one in which the genetic material has been changed in a way that does not happen naturally by mating and/or recombination. In contrast, a “Living Modified Organism” (LMO) does not contain foreign genes and is created through natural mutations or conventional breeding programs without human interference. The established international regulatory courses typically exclude genome-edited plants and animals, using novel biotechnological techniques, from GMO/LMO classification [29,30]. Accordingly, organisms generated by the GE techniques are excluded from the GMOs.

Concerns have also been voiced over transparency for consumers. So far, consumers have expressed very little interest in genetically engineered products. For instance, in Canada, the demand for transgenic salmon, which is expected to grow faster and be commercially viable, was so limited that the economic viability of AquaBounty (the company behind it) is at risk [23]. Several reasons for the slow progress in this formerly promising field include, the lack of public research financial support, regulatory hurdles, and concern about public opinion [23]. Added to the safety concerns are the raised ethical worries and welfare issues focused on animal breeding, especially in vertebrates, because hundreds of genetic transformation experiments are conducted in living embryos, causing the loss of embryos during impregnation and pregnancy [31]. In this context, it is worth mentioning that this type of research is costly. It is also true that both public sector and private funding is scanty, as compared with applied and basic biomedical research.

Several review articles about the obstacles to the widespread adoption of GE food animals have appeared in the published literature. The present review does not reiterate these matters but focuses on some of the new applications of GE. Also, it does not address genetically engineered farm animals developed for biomedical research applications. A new animal drug or nonfood species is reared and utilized in contained and controlled conditions, such as GE laboratory animals employed in research institutions.

2. Genome/Gene Editing Technologies

2.1. Past Technologies

Gene engineering techniques refer to processes that involve overexpressing or inhibiting a unique gene. It was reported that recombinant gene cloning was successfully achieved in 1972 (Figure 1) [3]. The first attempts at generating transgenic mice were carried out during the 1970s by exogenous gene transfer and germline transmission into a fertilized embryo (Figure 1) [32]. However, gene modification through viruses had disadvantages, including experimental safety concerns, undesired incorporation into the host genome, and possible oncogenic effects. The other methods of DNA delivery are predominantly microinjection and electroporation (Figure 2) [33,34,35].

To improve GEN technologies, HR was used in the year 1984, for more precise gene insertion. With HR, nucleotide sequences are switched between similar or identical DNA molecules. The SCNT [36] or microinjection (MI) (Figure 2) techniques is used to initiate the genome modifications for the construction of genetically engineered livestock. In an HR method, exogenous genes have to be transferred into somatic cells or in vivo fertilized embryos. Homologous recombination occurs when cells divide, and a cycle of checking processes is required over a long time for the selection of gene-engineered cell or embryo [37]. The technically complicated process of constructing GM animals involves three critical steps at early embryonic stages: isolation of zygotes from pregnant females, transferring nuclei into the separated zygotes, and subsequently implanting the modified embryos into pseudo-pregnant female recipients to obtain viable progeny.

Traditional breeding methods are based on phenotypic information. Unfortunately, long-term selection led to cell senescence and death in embryos and somatic cells [38]. Therefore, it wasn’t easy to efficiently generate genetically engineered livestock animals in the 1990s [39]. Research on animal production with the required trait through SCNT concentrates on animal production by constantly applying GE tools to SCNT, since it can decrease the aging of somatic cells [3]. The following paragraphs give descriptive examples from each corresponding field.

Before the advancement of SCNT in livestock in 1997, most genetically engineered animals were generated mainly by MI of DNA into the cytoplasm of the pro-nuclei of embryos (zygotes) (Figure 1). The first transgenic large animals were produced in 1985 by MI to improve the growth of agricultural livestock, and this allowed the successful production of genetically modified animals, including rabbits, goats, sheep, pigs, and cattle [40]. The initial stages of GE are followed by embryo transfer into a recipient. Direct DNA MI was the first practice that led to regular and relatively easy success in mammals [41]. The first GEN farm animals were constructed by MI of the genes of interest into the pronucleus of zygotes [42]. Microinjection was the first practical method for the generation of transgenic animals through the injection of an extraneous DNA construct into a fertilized ovum [43]. The construct is randomly incorporated into the recipient oocyte’s genome. Following this, the zygote resumes embryonic development and the embryo is translocated to a foster mother and ultimately grows into a transgenic animal [44].

The transgenic recombinant DNA (rDNA) integration levels were low, 10.4% and 1.3% in pigs and sheep, respectively, an issue that has continued to be problematic for livestock. Also, less than 1% of embryos are injected, and about 12% of farm animals are transgenic [39]. In addition, MI was unsuitable for use on farm animals, due to long gestation periods, small litter sizes, and reduced transgene integration rates. Furthermore, multiple transgene copies are integrated at random, since genes can only be added, not replaced or deleted. These conditions hinder the correct control of gene expression and interfere with endogenous gene function. Thus, large amounts of oocytes are needed to be injected because the overall efficacy of the process is very low [45].

In invertebrates [46] and lower vertebrates [46,47], pronuclei are not observable genes. Therefore, MI must be executed in the cytoplasm, using more significant quantities of DNA. For unclear reasons, the success rate of this method is quite variable depending on the species. Microinjection into a fertilized egg is the preferred method for GE in fish [46,47]. However, GE can also be made on fish cell lines. Genome editing utilizing an electric pulse to transfer the protein tools that execute the GE into cell lines from some fish species, including Atlantic salmon (Salmo salary), rainbow trout (Oncorhynchus mykiss), and Chinook salmon (Oncorhynchus tshawytscha) has been indicated to be highly successful [48,49]. Further, microinjection remains ineffective in the laboratory fish medaka, Xenopus, and chicken. As previously reported [50], the extraneous DNA usually does not incorporate into the genome of these species.

Initially, transgenesis via pronuclear MI suffered substantial limitations since targeted disruption was not promising using these approaches [20,51]. With MI, mosaicism is possible [52], which occurs when an organism has two or more genetically different cell populations, due to a genetic mutation that did not influence all the cells [53]. Thus, the desired genetic change is not found in all cells of GEN animals produced using zygote MI. Not every copy of the genome in every cell is modified; rather, only certain copies of the genome are altered. Therefore, animals resulting from modified embryos don’t generally have 100% altered cells. Some cells have changed DNA, and others have unaltered DNA. Mosaic animals may express the desired trait; however, whether or not they can pass it to their progeny depends on whether their sperm or egg cells possess the DNA changes. Mosaicism is particularly problematic in large livestock species having long reproductive cycles, as it can take years to generate non-mosaic homozygous progeny through crossbreeding [35]. Creating the desired phenotype coupled with germline transmission could require generating some transgenic founder lines, which will ensue by breeding. Mosaicism can prevent the organism from developing distinct observable traits and present challenges for breeding the wanted genetic alteration [19]. Furthermore, pronuclear injection could not regulate the integration site or the number of copies of the integrated genes.

The most profound restriction was that DNA can only be added but neither deleted nor modified in situ. In addition, the incorporation of exogenic DNA was random, resulting in erratic transgene expression due to the integration site effect. Moreover, random integration into the genome poses a danger of disrupting critical endogenous DNA sequences or stimulating cellular oncogenes, which could potentially harm the animal’s health [35].

Additionally, despite the widespread use of the MI method in the generation of edited animal models, it requires expensive micromanipulation equipment and expert personnel. This requirement introduces operator-dependent experimental differences as a confounding factor in editing experiments [54]. Moreover, it is time-consuming, which limits the number of microinjections that can be performed in each attempt [55].

To increase the efficacy of MI, lentivirus or retrovirus was inserted into a zygote, revealing higher efficiency than DNA injection. GEN has become considerably more efficient and precise since the emergence of GE tools such as ZFNs, TALENs, and CRISPR/Cas9. Furthermore, MI’s efficiency in constructing genetically engineered animals has significantly improved as well [56,57]. With the introduction of the ribonucleoprotein (RNP) complex in the CRISPR/Cas9 structure, electroporation, one of the most usual techniques for RNP delivery, has become feasible [5,58].

To deliver DNA sequences more significant than four kilobases, viruses carrying the DNA may be inserted in the electroporation technique, which utilizes an electric pulse to make pores in the fertilized egg [59], across which the DNA carrying the GE tools can pass [60,61,62]. The ‘foreign’ viral DNA integrates into the genome at minor frequencies (~0.1%), though its relative incidence within the developing egg cell drops with each round of cell division [63,64]. The electroporation is appropriate for inducing indel mutations, large deletions, and small insertions [65]. Consequently, different kinds of gene-engineered animals have been generated by GE tools, leading to improved productivity, disease resistance, and biomedicine improvement. Electroporation is an efficient method that has permitted the generation of gene-engineered animals, with an easier and faster approach than the previously laborious and expensive MI technique [5,54,65]. It can be concurrently applied to many zygotes and only requires a stereomicroscope, electroporator, and fusion chamber [54]. This approach allows for a more significant number of oocytes or zygotes per female receiver at a time and, finally, to achieve live offspring with targeted gene modifications [54,66]. Combining the mass production of In vitro fertilization (IVF) embryos with the GE by electroporation of gRNA/Cas9 RNPs, Cas9 mRNA, and gRNA strategy compensates for poor IVF outcomes with high editing frequency [54].

To overcome mosaicism and low efficacy associated with MI, SCNT was introduced and successfully used to generate cloned livestock. Before the advance of GE tools (ZFNs, TALENs, and CRISPR/Cas9), SCNT was favored over MI [5]. Since the 2000s, SCNT or cloning (Figure 2) has been the most widely used strategy for breeding genetically engineered or genome-edited farm animals like pigs [67], cows [67,68], and goats [69]. In addition, SCNT has been employed in generating genetically engineered animals with genetically engineered somatic cells [3,70]. This method is a protocol in which the nucleus of a somatic cell with a desired mutation is moved to the cytoplasm of an enucleated metaphase II stage oocyte, resulting in a new animal genetically identical to the somatic cell donor [71].

The production of GEN animals using site-specific modifications is possible at present. The SCNT technology was further found for other vital livestock species (Figure 1): cattle [72], goats [73], pigs [74,75], and sheep [17,76], offering the first cell-mediated stage for livestock GEN. The strategy is performed by injecting an exogenous gene and the targeted interruption of an endogenous gene [77], known to cause hyperacute refusal after organ transplantation. Many other GEN livestock models have been described [67,77].

Manipulating the preferred gene in somatic cells (usually fetal fibroblasts) is easier and more accurate than in embryos. To verify that the wanted genetic changes are present in the cells, single-cell-derived colonies are separated and checked for the presence of these changes. Next, the cells are donated for SCNT [67,76,78]. The genetic modifications hosted in the genome of somatic cells should be performed over to offspring by SCNT involving targeted modifications. Compared with the zygote manipulation method for GEN animal production, SCNT has several advantages, including a simple selection of donor cells with the wanted gene or mutation. Furthermore, since the whole animal originated from a single GEN donor nucleus, the risk of mosaicism is eliminated [75]. This cell-mediated GE strategy enables researchers to verify that the gene-edited cells contain the desired genetic modification before implantation in recipient mothers, ensuring successful animal production. GEN animals created by SCNT often need fewer recipient animals than the number of animals required for the zygote micromanipulations [76]. This approach reduces the occurrence of genetic mosaicism, reduces the time frame for generating the sought genotype, and decreases the overall cost of animal production [12]. Still, this approach is more technically demanding and typically has little incidence of term development. Due to low efficiency, only a few GEN animal models are registered. Since targeted modifications in somatic cells depend on the incidence of endogenic HR, their efficacy in these cells is extremely low [67,77]. The reduced effectiveness causes modifications of only one out of two alleles at a time. Since only heterozygous mutations can be established, breeding steps are necessary to produce genetically engineered (GEN) animals with homozygous modifications. The prolonged gestation period of some livestock species, particularly the large animals, adds days needed for finalizing breeding steps. The low rate of HR obligated the use of a selectable indicator, for instance, Neomycin, to select somatic cells holding the correct genetic modification [21,67,77].

In addition, potential cloning-associated epigenetic changes might add to the GEN animal phenotype. Thus, GEN animals generated through this procedure often carry a foreign gene that can improve antibiotic resistance in the animals. Despite these limitations, SCNT remains the primary method for constructing the KI gene-edited livestock [78]. Moreover, about 50% of the published KO farm animals were generated by SCNT.

Currently, SCNT allows gene KO and gene KI generation in farm animals [35]. The SCNT technology is being used to develop the poor fertility of buffalo, which are principal livestock in many countries. Although SCNT cloning of GE cells solved some of the problems associated with zygote micromanipulation, the low efficiency of generating live and healthy cloned progeny has limited the usefulness of this approach to produce GE livestock [79].

Moreover, the productivity of SCNT is poor [80], and animals born through SCNT often experience a high incidence of health problems such as abortions, mortality, and both pre- and post-birth abnormalities, raising animal welfare concerns [80,81,82]. The SCNT procedure is inefficient due to two main problems. Firstly, effective implantation has a set of logistical challenges. Specifically, foster mothers have to be biologically prepared for implantation [83]. Nevertheless, the success rate for implanting a fertilized egg or embryo is significantly lower than that of natural fertilization through mating. Second, the minimal levels of DNA methylation and elevated expression of the Xist gene in transgenic cloned calves. These conditions increase the incidence of stillborn female cloned buffalo relative to naturally breeding female buffaloes [84]. It has been reported that siRNA-mediated knock-down of the Xist gene successfully inhibits gene expression and stimulates the development of SCNT-cloned female buffalo embryos [45]. The possibility of GM animals being generated by SCNT was one of the powerful forces behind its development. The CD163 and CD1D KO transgenic pigs were produced using both SCNT and cytoplasmic injection into zygotes in vitro [85]. The targeting efficiency in both cases was 100%. Nevertheless, several technical hurdles limit the application of this method. First, the efficacy of cloning is very low in almost all kinds of farm animals (0.5–1.0%). Second, SCNT is accompanied by side effects such as aberrant reprogramming during embryogenesis and other developmental abnormalities in extraembryonic tissues of the produced animals, such as the placenta [86]. These abnormalities result in a low pregnancy frequency and higher instances of sudden death following delivery. In addition, anomalies such as obesity, immunodeficiency, and defects in the respiratory system are also seen in cloned animals after birth [87,88]. Therefore, the application of SCNT in agriculture has lagged primarily because of concerns about the time-consuming production process and the potential public criticism against the consumption of livestock, containing transgenes in the food industry. Several attempts have been made to improve SCNT efficiency, including addressing epigenetic factors to control reprogramming challenges. Two studies were devoted to injecting epigenetic factors throughout embryo development [89]. Yet, the aforementioned matters have not been fully resolved to date, and SCNT still has a reduced implantation rate compared to IVF, as reported lately [3].

Figure 3 outlines a protocol combining access at the embryo stage with genetic tools for producing genetically engineered livestock. SCNT necessitates the fusion of an enucleated egg cell acquired from the egg cell donor and a somatic cell resulting from the nucleus donor. Before this step, an exogenous gene is delivered to the somatic cell stage using methods such as virus, MI, or electroporation to produce a gene-engineered cell using one of the available techniques. After that, the developed engineered embryos are transferred to a foster mother to generate gene-engineered livestock.

2.2. Present Technologies

Over the past hundreds of years, traditional methods for selecting desired traits have proved to be slow, inaccurate, and insufficient to meet the rapidly growing global population demands. Without established ESC lines, SCNT had to be utilized to generate numerous genetically engineered livestock models. With the GEN by SCNT, genetically engineered livestock models could be produced by bringing site-specific modifications.

Although successful, only a few genetically modified livestock models have been developed due to low productivity and associated congenital disabilities. Owing to their precision, efficiency, and versatility, the modern GE systems ZFN, TALEN, CRISPR-Cas9, BE, and PE have emerged as cutting-edge biotechnological approaches [91] in animal research and sustainable food security. Compared to conventional breeding techniques (Table 1), notably crossbreeding and mutation breeding, these tools markedly decrease the time and effort required to establish a GEN animal. The emerging technological innovations in GE systems are “ideal” approaches that have improved efficiency in creating GE livestock models, altering a genomic sequence, and presenting higher DNA sequence precision with low or no off-target effects. Another advantage of using GE techniques is the application of direct inoculation into developing embryos to cause targeted mutations, consequently excluding side effects associated with SCNT.

Conventional genetic modification methods put genes at multiple and random locations in the genome, demanding researchers to decide which insertion is the most beneficial [92]. The application of conventional technologies to livestock breeding depends on the positive and negative selection of the favorite traits, and it usually needs generations to choose and stabilize genetic characteristics in a population. In contrast, the relatively modern technologies of GE offer a one-step generation of an animal with predetermined genetic and phenotypic characteristics based on the knowledge of gene functions [35,64]. The past GE techniques were developed by randomly inserting the desired gene into the host genome. Technically, random insertion has several possible disadvantages [93]. First, the targeted host gene integration site can only be identified after examining the transgenic animals by polymerase chain reaction (PCR) and DNA sequencing. Second, gene insertion can change the expression of endogenous genes. Third, several gene copies can be incorporated into the host genome. The random modification of the genome was ineffective and costly for animal breeding. Moreover, there were safety concerns about malfunctioning original genes or influences on the regulation of gene expression [93].

Due to their ability to precisely edit target loci, modern breeding techniques result in faster and more efficient livestock improvements than traditional breeding methods. However, the long-term consequences of GE are not fully known, and there are concerns that it may threaten animal welfare despite being less risky than classical GEN methods [5].

Present GE tools enable quick investigation of first-class traits, such as productivity and resilience to diseases and climate changes, without minor issues. The following sections discuss the basic principles of these present GE strategies and the major insights into their recent applications and achievements.

Basic Principles

The early gene targeting experiments relied on the spontaneous integration of exogenous DNA into the genome. However, the rate of successful genetic modification using this procedure is very low (1 in 1000,000 sites and 1 in 10,000 cells). As a new GEN technology, GE can precisely identify and adjust a definite sequence of a specific gene in an organism’s genome. The precision of these systems originates from the ability to identify particular DNA sequences on the genome and induce site-specific DSBs [94]. The DNA breaks lead to activation of the intrinsic DNA repair process to fix the DSBs since the DNA breaks may cause cell death if not corrected. The insertion of site-specific genomic modification is simplified by two major DNA repair pathways: NHEJ or HDR (Figure 4).

The error-prone nature of the NHEJ mechanism causes random base insertions or deletions (indels) and deletions of nucleotides at the sites of DSBs. This mechanism leads to gene KO as it often produces a frameshift of the amino acid codons and the development of a premature stop codon. On the other hand, if a donor DNA with homologous sequences to the target genome site is present, definite modifications at the nucleotide level can be introduced throughout the HDR pathway [97], In this process, the cell utilizes the sister chromatid of the injured DNA as a template for reparation. By integrating a designed donor template, particular modifications can be carried out at the DSB site through the HDR pathway [98]. In general, most cell types have seen a lower incidence of HDR than NHEJ. Whether the cells follow the NHEJ or HDR mechanism depends on the cell cycle, the make-up of the ends, and end resection [99].

A new BE platform, a C-to-G base editor (CGBE1), has been introduced [100]. The new system can distinguish specific sequences on the genome and initiate site-specific DNA breaks. This platform is the first CRISPR C-to-G base editor to present a transversion mutation (C→G) without a DSB. The latest GE tool is the PE, one of the most precise approaches for point mutation [101]. This novel protocol of PE has been designed to introduce point mutations with no donor DNA template for the HDR track or even the presence of a DSB in the target sequence (Table 2 and Figure 4).

Table 2:

Differences between advantages and disadvantages of ZFN, TALEN, CRISPR–Cas9, BE and PE gene-editing technologies.

Characteristic	ZFN	TALEN	CRISPR–Cas9	BE	PE
Endonuclease	Fok I	Fok I	Cas9	dCas	pegRNA
DNA binding system	Protein–DNA	Protein–DNA	RNA–DNA	R NA-DNA-Protein	R NA-DNA-Protein
DNA binding determinant	Zinc finger Protein	TALE protein	CrRNA/sgRNA	dCas/nCas	nCas9/pegRNA
Design and construction	Difficult	Moderate	Easy	Easy	Easy
Target fragment size/monomer	9–18 bp	14–20 bp	20–22 bp	4–8 bp	8–15 bp
Target recognition efficiency	High	High	High	Variable	Variable
Average mutation rate	Medium	Low	Low	Very high	Very high
Precision	Low	Moderate	Precise	More precise	Most precise
In vivo delivery	Easy	Difficult	Moderate	Difficult	Difficult
Multiplexing ability	No	No	Yes	*	*
Off-target effect	High	Lower	Variable	Low	Very low
Cytotoxicity	High	Lower	Low	*	*
Cost	Very high	High	Low	Very high	Very high
Cytotoxicity	High	Intermediate	Low	*	*
Time needed	Time-consuming	Time-consuming	Less time needed	Time-saving	Time-saving
Application	Not widely used	Widely used	Most widely used	*	*
Mechanism of action	DSBs with either NHEJ or HDR	DSBs with either NHEJ or HDR	DSBs with either NHEJ or HDR	DSBs with either NHEJ or HDR	No DSBs. Instead of replacing a base, it modifies the base chemical structure into another one

Abbreviations: BE: Base editor; bp: Base pair; CRISPR: Clustered regularly interspaced short palindromic repeats; Cas9: CRISPR associated protein system 9; CrRNA: CRISPR RNA; dCas: Catalytically inactive (dead) Cas; nCas: Nickase Cas; pegRNA: Prime editing guide RNA; PE: Prime editor; sgRNA: single-guide RNA; TALEN: transcription activator-like effector nucleases; ZFN: zinc finger nuclease. * Not evaluated. Source: [5,10,102,103,104].

Table 2 compares and summarizes the advantages and disadvantages of the GE techniques available for application in livestock research. The CRISPR/Cas9 system has reached unprecedented levels of efficacy for gene inactivation in almost all species. Notably, it enables the concurrent inactivation of multiple genes by utilizing more than a single target-specific guide RNA.

Through sequential nuclear transfer, one allele of gene A can be inactivated, followed by somatic cell nuclear transfer (SCNT) and re-isolation of somatic cells. Subsequently, the second allele of gene A can be targeted, followed by another round of SCNT and the isolation of cells to focus on gene B. If the genome-edited pig is produced by SCNT, followed by a simple selection of the edited cells by magnetic beads, it can score efficiencies of >90% for multi-gene inactivation [105]. CRISPR system was first applied to the mammalian genome in 2013 [106], and after that, it extended to a broad collection of cell lines and fish, bird, and mammalian groups, including livestock. This new GE system revolutionized the construction of GM animals. It enables researchers to modify genetic makeup that leads to improvements in livestock traits and construct more improved large animal models of human diseases. The pig recorded the maximum number of concurrently inactivated gene copies: 25 copies of porcine endogenic retroviruses [107].

Progresses in CRISPR/Cas9 GE have appreciably improved the ability to accurately disrupt genes and/or produce particular mutations by direct zygote handling (pronuclear or cytoplasmic injection or electroporation) [108,109]. Electroporation has been reported to be highly productive in creating indel mutations in bovine and porcine zygotes [110]. This method significantly simplifies the production of genetically engineered livestock, as it does not need micromanipulation expertise. Yet, genetic mosaicism remains a major problem using the zygote manipulation method [111].

As in the past GE techniques, which resulted in mosaicism [112], mosaicism occurs when DNA replication occurs before CRISPR-mediated genome editing, significantly reducing the possibilities for direct KO generation. The influence of mosaicism could be even more overwhelming if both somatic and germline mosaicism were found in the offspring. One of the strategies suggested for reducing genetic mosaicism is a transfer of CRISPR/Cas9 into either metaphase II (MII) oocyte or a very early zygote stage [113].

Nevertheless, the injection of CRISPR/Cas9 into MII oocytes did not decrease mosaicism compared to the zygote injection in sheep [114] and cattle [115]. The inability of CRISPR to identify its target site before some level of chromatin decondensation took place could cause these somewhat surprising results [12]. Another possible tactic for lowering mosaicism is shortening the longevity of Cas9 by speeding up its degradation [8]. This strategy can be achieved by marking Cas9 with ubiquitin-proteasomal degeneration signals mediating Cas9 degradation. Otherwise, the nuclear transfer technique using GEN cells could eradicate the threat of mosaicism. Recent studies showed that the microinjection of CRISPR/Cas9 constituents into swinish germinal vesicle oocytes efficiently reduces the risk of mosaicism [116]. About 83% of the mutated embryos were non-mosaic [116].

CRISPR/Cas9 system-mediated GE strategy has been used widely in studies involving the genetic breeding of animals. However, the system’s efficacy at inserting foreign genes is insufficient, mainly due to specific off-target effects [104]. CRISPR)/Cas9 technology is unsuitable for use in the GE of large livestock. New target GE systems, BE systems, have been developed using CRISPR/Cas systems [90]. The first BEs were created in 2017 [117], and the BE technique was the first innovation in the GE field after CRISPR/Cas9, because of the ability to complete precise point-mutation without a DSB. Unlike traditional CRISPR/Cas systems, the new system does not depend on cellular repair, employing the NHEJ or HDR mechanism. In contrast to DSB-mediated mutation, BE-mediated mutation is more precisely controllable. The advent of the BE system offers a powerful tool for accurate GE.

The principle of the BE systems relies on combining a nucleotide deaminase and unnaturally modified nCas9 protein (D10A nickase) to replace bases at the target location by creating a single cut in the double-strand. The latter is a single-strand break (SSB) instead of a DSB, as the D10A mutation in the RuvC domain alters Cas9 into a DNA nickase [118]. Generating an SSB instead of a DSB escapes the formation of indels [100]. According to different base modification enzymes, the BE systems consist of cytosine base editors (CBE) and adenine base editors (ABE). These changes can achieve C–G to T–A and A–T to G–C edits, respectively [119].

The application of ABEs overcomes the constrictions of CBEs, which can only edit C or G bases, and gives a wider range of base transformation choices. Unlike CBEs, ABEs do not need to stop the activity of alkyl adenine DNA glycosylase (AAG) [120,121]. The optimized CBE system was first efficiently utilized to produce base-edited pigs to obtain larger animal models [122]. Subsequently, many research groups have deployed CBE and ABE to KO various mammalian genes, for example, GGTA, MSTN, CD163, GHR, and others, for the improvement of rabbit and pig embryos [123,124], and human cells [122,125,126]. These results also suggest that the BE systems can improve livestock traits like reproduction, milk, and wool production.

In 2019, PE was introduced as a tool that can achieve targeted editing, including all 12 possible combinations of base-to-base conversions, insertions, and deletions, without the need for the template of the donor genetic material [101,117]. While PE represents meaningful promising progress in GE, this technology is still in its early stages, and further research and investigations are needed to unlock its competencies and potential. Prime editing is a catalytically inactivated nCas9 (H840A) that is fused with a reverse transcriptase (RT-nCas9) with the capacity to be transfected along with a prime editing guide RNA (pegRNA). The molecular mechanism of PE involves identifying and attaching the primer-binding site (PBS) of the target to the 5′ region of the primer-editing guided RNA (PegRNA), thus exposing non-complementary strands. Cas9 nickase (nCas9) cuts the ssDNA complementary to the template RNA via the RuvC nuclease domain, attaching primers for RT enzymes to nCas9. The PegRNA serving as the gRNA identifies the target DNA to achieve base-to-base editing [127,128]. These new procedures have a low chance of missing their target, and no new functional alterations will occur. Furthermore, base editing (BE)-mediated gene editing is not reliant on double-strand breaks (DSBs) DSBs-induced NHEJ repair pathway is random and may cause unnecessary indels production. In addition, excessive DSBs cause cytotoxicity. On the other hand, researchers also are not required to screen for very active sgRNA and Cas nucleases. Also, the BE system does not need donor DNA, a crucial part of the HDR repair pathway. Designing practical donor DNA for livestock and delivering donor DNA effectively into livestock cells are practical challenges [129].

The PE has been practiced in mouse cells [mouse neuro-2a (N2a) cells], of which the prime editor 3 (PE3) facilitated base transversion at three target locations of Hoxd13 and androgen receptor genes with an effectiveness rate between 8% and 40% [124]. Additionally, zygote MI of pegRNAs, targeting the same Hoxd13 gene, resulted in successful conversion mutations. The conversions G-to-C and G-to-T were obtained in 44% and 75% of blastocysts, respectively, with mutation incidences varying from 1.1 to 18.5% in each embryo. Although PEs are notably versatile tools for precise GE and have distinct advantages, they show lower efficiency than other GE strategies [130].

3. Applications of Genome Editing in Animals

In agriculture, GE is used to improve the characteristics of crops and livestock, such as enhancing their resistance to pests and diseases or improving their nutrient content. Genetic development of economically important traits is a key topic of livestock research. The continued development and refinement of GE technologies are anticipated to have a profound impact on various industries, with the potential to drive significant breakthroughs across multiple fields in agriculture. The following studies have been carried out to increase production based on farm animal species (Figure 5).

3.1. Gene Editing in Farm Animals

Breeding farm animals has become a primary focus in the field of animal breeding. In the last 10 years, GE technology has been used to breed various farm animals, including fish, chickens, rabbits, pigs, cattle, sheep, goats, and other species [10,90]. Genome editing technologies hold meaningful promise for reforming agriculture by allowing precise and effective genetic modifications in livestock. Using Cas9 to insert favorable genes into animals accurately, scientists can support the industry’s healthy and effective development, cut economic losses, and guarantee high food safety standards. Recent investigations suggest that the GE systems can improve effectively animal traits like growth and reproduction as well as production of egg, milk, and wool [56,131,132].

In aquaculture species, the genes associated with main traits, such as growth, disease resistance, and robustness, are still very inadequate, which impedes the CRISPR/ Cas applications in this field [9]. CRISPR/Cas assembly needs to be transported into fertilized eggs at the one-cell stage in fish [47,133]. The time required for the progress of the zygote to first cell division differs from one fish species to another; thus, it is essential to know this timing for successful GE. Microinjection of many eggs quickly is labor intensive and requires an expensive specific MI platform. Additionally, access to newly fertilized embryos is difficult for species such as certain shrimp. Consequently, the substitute methods of CRISPR/Cas9 delivery have to be advanced and examined for breeding purposes, for instance, electroporation, the MI of unfertilized ova, sperm-mediated transfer, or editing PGCs [9].

3.1.1. Breeding of Improved Characteristics

Table 3 shows examples of GE applications for fish, poultry, and livestock production trait perfections. The main interest fields covered under this umbrella include reproductive performance, egg and meat production, and improvements in milk quality. However, it should be remembered that the full KO of a gene that negatively controls growth may lead to welfare worries. These concerns involve miscarriage, difficulties during birth, developmental abnormalities, or even offspring mortality, as observed in pigs [134], as well as in cattle [135], goats [136], and rabbits [136]. By contrast, spontaneous changes in this gene that have happened naturally are not substantially associated with such extreme alterations in muscle mass. Avoiding this using GE will likely need more accurate mutations in this gene [137].

Breeding in Fish

The aquaculture industry provides potential promises to meet the demand for high-quality food as it offers nutritionally rich, affordable supplies [138]. The first fish species to be exposed to an organized family-based SB program began in 1975. These programs have generated genetically discrete lines of fast-growing salmon. However, since the 1970s, millions of these fertile farmed salmon have leaked into the wild [139]. These sea cage escapees are a significant threat to the fish farm business. For example, mating between wild salmon and domestic salmon escapees endangers the genetic integrity of the wild salmon [138]. Using the CRISPR/Cas9 system, researchers KO the dead-end (dnd) gene in novel sterility models in Atlantic salmon (Salmo salar), resulting in a loss-of-function mutation that prevents germ cell differentiation [50,140] (Table 3). The resulting mutation prevents sexual maturity, leading to a sterile phenotype and consequently protecting the integrity of the wild-type population. Subsequently, dnd KO individuals have increased growth, higher flesh quality, and lesser susceptibility to infectious diseases [49,140]. Germ-cell-free Atlantic salmon stayed immature and did not experience puberty [141]. In another investigation, sterile channel catfish (Ictalurus punctatus) were developed by editing the luteinizing hormone (LH) gene utilizing a modified ZNF technology with electroporation [142]. In a similar study, scientists at Kyushu University used platinum TALEN to KO the LH gene in Japanese anchovy (Engraulis japonicus) [8].

Myostatin (MSTN; previously called GDF-8) gene is extraordinarily well conserved in many different species and is expressed almost exclusively in skeletal muscles [10,12]. The gene assembles the transforming growth factor-beta superfamily, which harms skeletal muscle growth. Inhibition of MSTN benefits aquaculture as it creates fish with improved development and increased muscle mass [143]. As shown in Table 3, CRISPR tools have been utilized for growth improvement by deactivating the MSTN gene in many fishes such as the common carp (Cyprinus carpio) [144], channel catfish (Ictalurus punctatus) [145], Red Sea bream (Pagrus major) and tiger puffer (Takifugu rubripes) [146], as well as olive founder (Paralichthys olivaceus) [121], Pacific oyster (Crassostrea gigas) [147], blunt snout bream (Megalobrama amblycephala) [148], and Nile tilapia (Oreochromis niloticus) [28]. The MSTN KO Red Sea bream was reported to significantly enlarge its muscle mass (16% increase of skeletal muscle), decrease body length, and better feed efficacy, leading to better overall growth, which was not seen in other fishes [146,149]. Furthermore, the CRISPR/Cas9 strategy was utilized for MSTN-targeted interference to obtain the first growing mud loach (Misgurnus anguillicaudatus) [150]. Aside from the MSTN gene, CRISPR was also used to improve growth-associated characteristics in the tiger puffer (T. rub ripe) by disturbing the leptin receptor gene that regulates appetite, making fish eat more [146,151].

Breeding in Poultry

Chicken is a key animal species globally; thus, genome-tailored chickens will spread avian biology and be valuable for agricultural and industrial applications. Genome editing technology can develop avian models for different applications. However, the generation of gene-edited poultry using various approaches, including CRISPR–Cas9, has been less fruitful than for mammals. It is more challenging due to the complex structure of the fertilized bird egg [10,152]. The application of genetic techniques established in mammals to bird species is impeded by the difficulty of getting into the fertilized oocyte nucleus and handling avian oocytes [153]. Since poultry has unique reproductive features, the lack of ESC continues in poultry species, and the traditional SCNT technology utilized in cloning mammals cannot be used in poultry. In chickens, genetic information to offspring is carried by the PGCs, and both donor and recipient PGCs grow concurrently in the recipient testes, resulting in the rejection of the donor PGC and a low sperm count [154]. Although PGC is an excellent tool for precise genome adjustments, only chicken PGC can be reliably cultured in vitro [155]. Using germline-competent chicken PGCs, which can be preserved in culture with no loss of germ cell properties, it is possible to produce particular gene-tailored chickens without genomic integration of foreign transgenes [155]. A primordial germ cell-mediated germline transfer system has been established to produce gene-edited chickens [153,155]. Despite being possible, the procedure’s success largely relies on the quality of PGCs, which could be inconsistent. Due to its low efficiency, the first knockout (KO) chicken was not produced until 2013 [156].

Together with in vitro culture and manipulation of chicken PGCs, ovalbumin-KO chickens were produced using TALEN [157] (Table 3). In the chicken egg white, total proteins account for approximately 11% of the egg’s weight. Ovalbumin (OVA) constitutes about 54% of whole egg white proteins [157]. Due to its richness in protein content, the chicken egg is the best system for constructing bioactive materials. However, too much OVA obstructs the purification of biofunctional proteins. Consequently, adjusting the egg-white composition would allow for agricultural and industrial uses [157]. Using a CRISPR/Cas9-mediated KI system, bioreactor chickens for human interferon β (hIFN-β) were produced [158]. They inserted the hIFN-β gene into a precise locus of the chicken OVA gene. That study proved the possibility of a CRISPR/Cas9-mediated KI transgenic system at the chicken OVA locus leading to elevated-level production of bioactive biomaterials. However, producing KO birds employing the CRISPR-Cas9 system is technically challenging due to avian and mammalian species’ developmental and physiological differences [157]. The G0/G1 changeover gene 2 (G0S2)-edited chickens was constructed using the CRISPR-Cas9 system [158]. The G0S2 protein is a repressor of adipose triglyceride lipase (ATGL, also called patatin-like phospholipase domain-having protein 2), activating the initial lipolysis reaction [158]. It can be inferred that the consistent reactivity of the ATGL enzyme, resulting from the removal of G0S2 suppression, would lead to the continuous hydrolysis of triacylglycerols in adipose tissue, thereby reducing lipid content and fat accumulation.

Additionally, researchers discovered a phenotype of decreased fat deposition in the abdomen of the G0S2 KO chickens, which may be a possible target gene for trait development [159]. Notably, the mutated animals have been found to survive and reproduce normally [10]. Too much body fat in broiler chickens frequently causes leg weakness. Other problems, such as diminished reproductive performance, have been reported [160].

An MSTN chicken model using D10ACas9 has been effectively constructed [155] (Table 3). The skeletal muscles of the chest and the leg of MSTN mutant chickens were extensively expanded due to myofiber hyperplasia and hypertrophy [155]. Furthermore, compared to the corresponding wild types, these MSTN mutant chickens had significantly less fat deposition in their abdomens [155]. CRISPR recombinant adenovirus was injected into the EGK stage XI Japanese quail (Coturnix japonica) blastoderm [161]. MSTN mutant quails with improved feed significantly in quail with targeted MSTN gene were obtained. Further examination showed a higher feed conversion rate in the MSTN mutant quails [161].

Investigating the combination of PGC using CRISPR–Cas9 for poultry GE has more potential for a scientific breakthrough. Improved meat production performance was achieved in livestock by injecting MSTN–KO sgRNAs’ adenovirus vectors into chicks’ leg muscles [162]. Although MSTN expression was significantly downregulated, gene-edited offspring were still not bred.

Breeding in Pigs

The ZFN technique was fruitfully used to KO the exogenous enhanced Green Fluorescent Protein (eGFP) gene in porcine somatic cells, proving that the ZFN-KO system might be applied to domestic animals [163]. The first ZFN-mediated KO pig of an enhanced GFP transgene was bred in 2011 [164]. Mutations in the MSTN gene were targeted by ZFNs to generate a double-muscled phenotype in Meishan pigs (Sus scrofa domesticus) [165] (Table 3).

Based on the advantages of TALEN, many researchers have started to apply TALEN procedures to modify and improve livestock genomes, proving its excellent potential for application. It was reported that 64% of TALENs had high action in primary porcine cells [166]. Furthermore, direct injection of TALEN mRNA into zygotes of livestock resulted in the KO of target genes in about 75% of embryos (29% in swine). Also, TALENs successfully resulted in MSTN-KO in porcine somatic cells and the generation of KO swine with significant changes in muscle phenotypes (Table 3).

Pigs were the first large farmed animals edited using CRISPR-Cas9. Researchers [167,168,169,170,171] have used CRISPR and other gene editing procedures (Table 3). They successfully edited the MSTN gene in both local Chinese pig breeds and commercial Large White pigs, creating MSTN knockout (KO) pigs with an increased lean meat percentage. In addition to having comparatively high muscle mass, all pigs with MSTN mutation also displayed improved productivity. However, editing the third exon of MSTN in commercial Large White pigs led to a reduced survival rate and limb abnormality of piglets after birth [134,170], though targeting the first exon could efficiently avoid the deformities reported with the third exon GE. Another research group [165,167] found that homozygous MSTN KO Meishan pigs and Erhualian pigs did not have congenital disabilities.

Another likely candidate gene for refining meat assembly in livestock and developing curative medication for muscle disorders is the FBXO40 protein-coding gene, whose expression is confined to muscle. FBXO40 KO pigs generated by CRISPR/Cas9 have been made recently but showed only marginal amplification in muscle mass (4%) compared to controls [172]. Interestingly, the KO pigs grew usually, with no pathological signs found in major organs. In addition, CRISPR-Cas9 was used to knock the non-coding region of insulin-like growth factor 2 (IGF2) into Bama pigs [173]. They reported that the production of excellent meat quality did not change the health of the pigs. The CRISPR–Cas9 was used to knock mitochondrial uncoupling protein 1 (UCP1), which regulates and maintains the body temperature into piglet chromosomes to farm cold-resistant pigs with healthy lean with low fat [174].

Researchers [175] have reported a G to A mutation at position 3072 in the third intron of the porcine IGF2 gene, which affects skeletal muscle growth, fat deposition, cell proliferation, and differentiation. This mutation prevents the binding of transcription repressor ZBED6, improving the expression of the IGF2 gene and thus increasing the growth rate [175]. Such preferred mutations are commonly found in foreign marketable breeds but rarely in Chinese pig breeds. The binding sequence of ZBED6 in the IGF2 gene locus was deleted in 2018 [173]. This work was accomplished by MI of Cas9 nickase mRNA and a pair of sgRNAs into swinish zygotes. As a result, the expression level of the IGF2 gene was improved, and, therefore, the meat production of Bama pigs significantly increased. Using the CRISPR system, the ZBED6 binding sequence was disrupted in porcine fetal fibroblasts [176]. After that, they combined the SCNT technology and obtained miniature spotted pigs in Liang Guang with high lean meat levels. The gene FBXO40 is another possible candidate gene for the development of meat production in livestock, which is specifically expressed in muscles. CRISPR was used successfully to generate FBXO40 gene KO pigs, and the muscle weight of the genetically engineered pigs increased by about 4% compared to the wild-type control [172].

Finally, a one-step method that joined the HA3A-BE3-Y130F BE with the porcine zygote injection produced concurrent gene mutations in CD163, MSTN, and IGF2 [126]. The analyses indicated that the expression degree of CD163 and MSTN in the triple gene-edited pigs declined while the expression level of IGF2 improved, as designed. Interestingly, the growth performance and disease resistance were positively developed.

The analyses indicated that the expression degree of CD163 and MSTN in the triple gene-edited pigs declined while the expression level of IGF2 improved, as designed. Interestingly, the growth performance as well as the disease resistance were positively developed.

Breeding in Cattle

Cattle are acknowledged as the most incredible animals for producing significant amounts of meat and/or milk, making them an important source of protein [22]. However, cattle have low fecundity and a long generation interval compared to other livestock species, which slows genetic progress [137]. Yet, the dairy industry was well-positioned for the fast adoption of gene deletion. Beta-lactoglobulin (BLG) is a key milk allergen found in goat and cow milk but not found in human milk. Neither fermentation nor heat treatment can remove BLG from milk [177]. ZFNs GE and SCNT technology solved this long-standing problem by constructing a cow that lacked the protein BLG [178] (Table 3). Subsequently, cow milk was “humanized” using ZFNs and SCNT to insert human milk protein, Lactoferrin or Lactalbumin, into the endogenic BLG gene region [178]. The BLG gene KO cows were structured from fertilized eggs to expand the market for milk and its derivatives [179]. This gene can be KO through the CRISPR/Cas9 technique to generate hypoallergenic cow milk [180].

MSTN biallelic mutations in cattle were efficiently generated using ZFNs to improve meat production [181]. Direct injection of TALEN mRNA into zygotes of livestock resulted in the KO of target genes in about 75% of embryos (43–75% in bovines). In 2015, the MSTN gene was edited in cattle using TALENs and SCNT to produce high-quality meat [135] (Table 3). These experiments resulted in the successful birth of cattle with better muscle mass. Generation of MSTN-mutated cattle without exogenous gene insertion using CRISPR-Cas9 in vitro fertilized embryos has been published [5]. This research team transmitted MSTN KO germline into cattle via CRISPR-Cas9. They successfully produced beef cattle with higher muscle mass. These GE outcomes in cattle proved that the MSTN mutation is passed on to the next generation via the germline, laying the basis for the mass construction of MSTN mutation cattle.

CRISPR-Cas9 was used to KO the OCT4 gene, which plays a vital role in maintaining the pluripotency of mammals’ early embryonic stem cells [182]. Improved electroporation technology was used to deliver CRISPR-Cas9 targeting the sgRNA-Cas9 protein complex of OCT4 into fertilized bovine eggs, decreasing operational difficulties and producing breeding transgenic animals easier [183]. These data offer an animal model for handling early human embryonic growth-related defects and assist further research into enhancing cattle reproduction.

Another important field in modern livestock for the application of GE technology is the cultivation of hornless cattle to reduce the dangers of possessing horns for the animals and the farmers or animal caretakers [103,160,184]. Therefore, dehorning of cattle is a demanding action to avoid accidental injury. Traditional means for physically removing animal horns are costly and painful, and this action raises animal welfare concerns. Using natural selection and breeding in naturally polled cattle breeds to produce polled livestock can take several generations to show effect. Using a TALEN-based GE approach, the genome makeup of the horn-free Holstein cow was changed by the polled (hornless) locus. After that, polled-edited cattle were generated following a nuclear transfer of gene-edited somatic cells. Later on, CRISPR–Cas9 was used to transfer a gene that controls dehorned traits into the genomes of fibroblasts derived into the genomes of fibroblasts originated from horned Mongolian cattle [185]. Furthermore, the Polled gene was isolated and integrated into the genome of fibroblasts extracted from a horned bull using the CRISPR/Cas12a system, followed by somatic cell nuclear transfer (SCNT) [186] (Table 3).

The black-coated Holstein dairy cows are prone to high-heat stress due to absorbing much light. Heat stress substantially negatively impacts growth, reproduction, and milk production [112]. In this context, a critical GE application was the induction of a mutation in the Pre-melanosome protein (PMEL), a gene that controls the black color in the coat; using CRISPR-Cas9, gray-coated mutated calves were successfully born [187,188]. Another method to mitigate heat stress in Holstein cattle is by editing the Slick hair coat (SLICK) locus located in the Prolactin hormone receptor (PRLR) gene, which determines hair length [189]. Animals with SLICK haplotypes possess short, fine hair and can handle heat stress. The PRLR gene’s exon 11 (SLICK2) region, which is well adjusted to hot climatic situations, has a mutation (C > T) in Limonero and Carora cattle [190]. Consequently, inserting this (SLICK) mutation into the PRLR gene of heat-sensitive breeds reduces heat stress in animals subjected to hot and humid weather conditions [188]. Interestingly, the cows with SLICK gene mutations have very low health risks.

Breeding in Sheep and Goats

Sheep and goats are essential livestock in farming production and play a vital role in animal gardening. For goats and sheep, scientists use GE technology to modify different species’ traits: growth status, fertility, milk production, and cashmere yield [104].

It is known that the fecundity (Fec) family of genes is linked with the fecundity of sheep. CRISPR-Cas9 proved to be an effective editing strategy for developing superior traits in farm animals, boosting the progress of animal husbandry [191]. The Booroola fecundity (FecB) homozygous mutant sheep were bred using CRISPR–Cas9 by transferring ssFecB DNA oligonucleotides into the zygote of sheep (Table 3). The resulting ewes had superior lambing rates and better reproductive rates. The lambing rate was also improved by a simultaneous edition of the Hyaluronidase 2 (HYAL2) and Ovine prion Protein (OPrP) genes using CRISPR-Cas9 [192]. In this context, efficiently generated goats were obtained by introducing defined point mutation (I397V) in goat growth differentiation factor 9 (GDF9) through CRISPR/Cas9 [193]. It was found that this technique can enhance the ovulation rate and increase the litter size. Further, the Aralkylamine N-acetyltransferase (AANAT) gene was microinjected into unfrozen and frozen sheep embryos [194]. The results indicated no significant difference in the reproductive capabilities of transgenic offspring between the two environments, however, AANAT transgenic animals had good reproductive potential.

The use of SCNT and TALENs in sheep was found to have increased muscle production phenotype [195]. Previous reports [196] revealed that TALEN pairs could effectively induce KO of target genes in numerous livestock, with a 20–60% knockout efficacy. Amazingly, TALEN-mediated gene KI effectiveness is greater than 30% at some loci [196].

Also, in sheep, studies used CRISPR/Cas9 technology to generate sheep with the MSTN KO gene [56,129,196,197,198]. The same strategy was efficiently employed to increase meat production in goats [136]. Targeted MSTN was inserted into the cytoplasm of prokaryotic embryos to produce gene-edited goats with dual muscle features [196]. Moreover, it was confirmed that the loss of MSTN function in sheep skeletal muscle satellite cells (sSMSCs) enhances the differentiation and growth of sSMSCs [199]. Single-nucleotide point mutations in the Recombinant Suppressors of Cytokine Signaling 2 (SOCS2) were induced using CRISPR–Cas9, increasing sheep weight and milk production. More recently, the MSTN bialleles KO had many mutations without affecting meat quality [200]. A double-allele and a single-allele MSTN gene-mutated goat were bred through the microinjection of fertilized eggs [201]. Fetuses of both edited sheep had “double muscle” and substantially increased weight and length.

As for milk, researchers have improved the constitution of sheep milk by genetically amending essential nutrients and knocking out non-essential proteins. For example, goat milk is rich in protein and fat, and its composition is comparable to human milk. However, β- BLG is not found in human milk. The β- BLG is a key nonhuman milk allergen prone to sensitization [104]. Interestingly, the KO BLG gene was followed by human lactoferrin gene KI using TALENs in goats [202]. This elegant work indicated that GE, through TALEN-mediated HDR, could make genetically engineered livestock. Also, it can be utilized as mammary gland bioreactors to generate specific products efficiently. The advent of CRISPR–Cas9 was crucial to producing non-artificial milk or dairy byproducts, with low-fat or rich in particular nutrients or pharmaceutical ingredients [203].

It was discovered that using CRISPR–Cas9 KO BLG in goats cuts the protein amounts in BLG, laying the basis for improving the makeup of goat milk [204] (Table 3). The latter study recorded declined levels of fat, protein, lactose, and solid not fat in the milk by 5.5, 7.7, 8.0, and 7.7%, respectively. To improve the nutritional value of milk, CRISPR–Cas9 was utilized to KO components that affect milk characteristics, such as Stearoyl CoA Desaturase 1 (SCD1) [205] and Acetyl CoA acyltransferase 2 (ACAA2) [206]. CRISPR-Cas9 was used to transfer the AANAT and Acetylserotonin O-methyltransferase (ASMT) genes, which regulate melatonin expression, into the cytoplasm of fertilized sheep eggs, resulting in the creation of an AANAT/ASMT breast bioreactor [203]. The resulting genetically edited sheep had melatonin-rich milk. Also, the BLG gene has been KO by employing the CRISPR/Cas9 tool to construct hypoallergenic goat milk [207].

Cashmere goats are the principal local economic crucial livestock raised in Inner Mongolia. Cashmere is a significant natural raw material for the textile industry and, thus, an important source of income for farmers and herders. Researchers used CRISPR–Cas9 to stop Fibroblast Growth Factor 5 (FGF5), a secreted signaling protein that stops hair growth by inhibiting dermal papilla cell activation. This protein is the causative underlying the angora phenotype (long hair coat) [12]. Expression of FGF5 improved cashmere density and length in cashmere-producing goats [208] and sheep [131].

The Gene-edited Cashmere goats resulting from the KO of the FGF5 gene in Shaanbei white were followed in Cashmere goats using CRISPR–Cas9 [208] (Table 3). Compared with wild-type goats, significant increases in the number, density, and diameters of the secondary hair follicles in the skin of FGF5 mutant goats, as did cashmere length, were recorded [208]. Further studies confirmed the reported improvement of cashmere yield in sheep [131]. Cashmere goats carrying a mutant Ectodysplasin receptor-A (EDAR) gene, which inhibits hair follicle growth and development, were generated using CRISPR-Cas9-mediated somatic cell nuclear transfer (SCNT) to induce extracellular receptor abnormalities [209]. The SCNT technology and CRISPR–Cas9 were joined to produce EDAR gene-targeted Cashmere goats, which showed baldness [209]. Using CRISPR–Cas9 [208]. Compared with wild-type goats, significant increases in the number, density, and diameters of the secondary hair follicles in the skin of FGF5 mutant goats, as did Cashmere length, were recorded [208]. Further studies confirmed the reported improvement of cashmere yield in sheep [131]. Cashmere goats carrying an Ectodysplasin receptor-A (EDAR) gene mutant with inhibited hair follicle growth and development were generated using CRISPR–Cas9-SCNT-mediated GE extracellular receptor abnormalities [209]. using CRISPR–Cas9 [208].

Also, adding the exogenic thymosin β 4 (Tβ4) targeted incorporation into Cashmere goats stimulated hair follicle development, quickened the differentiation of hair follicle stem cells, and improved hair assembly [210]. CRISPR–Cas9 can disrupt genes that prevent hair follicle development into a perfect phenotype. This new knowledge would offer a scientific foundation for additional breeding of high-quality and high-yield Cashmere goat strains [104]. It would also provide insights into the therapy of androgenic alopecia.

In several investigations, two or three genes were targeted concurrently, resulting in double or triple gene KOs [208]). For example, to increase goat meat production and Cashmere yield harvest, Cas9 mRNA and sgRNAs targeting MSTN and FGF5 KO genes were injected into goat zygotes [211]. The KO efficacy of MSTN and FGF5 in 98 experimented animals was 15 and 21%, respectively. There was 10% of the goats had double-gene KO. Three GE sheep were obtained by targeting MSTN, ASIP, and BCO2 genes utilizing CRISPR/Cas9 and zygote microinjection protocols [211].

Table 3:

Examples of transgenic farmed animals produced using gene editing strategies for improved characteristics.

Animal	Breed/Common name	Scientific Name	Editing Tool	Edited Gene Abbreviation *	Trait Targeted	Reference
Fish	Atlantic Salmon	Salmo salar	CRISPR	dnd	Sexual maturity	[50,140]
	Channel Catfish	Ictalurus punctatus	ZNF+ electroporation	LH	Fertility	[142] [145]
		Ictalurus punctatus	CRISPR	MSTN	Muscle growth
	Japanese Anchovy	Engraulis japonicus	TALEN	LH	Fertility	[8]
	Common Carp	Cyprinus carpio	CRISPR	MSTN	Skeletal muscle growth	[144]
	Red Sea Bream	Pagrus major	CRISPR	MSTN	Skeletal muscle growth	[146]
	Tiger Puffer	Takifugu rubripes	CRISPR	MSTN	Skeletal muscle growth	[146]
		Takifugu rubripes	CRISPR	LPR	Appetite regulation	[146,151]
	Olive Founder	Paralichthys olivaceus	CRISPR	MSTN	Skeletal muscle growth	[121]
	Pacific Oyster	Crassostrea gigas	CRISPR	MSTN	Skeletal muscle growth	[147]
	Blunt Snout Bream	Megalobrama amblycephala	CRISPR	MSTN	Skeletal muscle e growth	[148]
	Nile Tilapia	Oreochromis niloticus	CRISPR	MSTN	Skeletal muscle growth	[28]
	Mud Loach	Misgurnus anguillicaudatus	CRISPR	MSTN	Skeletal muscle growth	[150]
Poultry	White Leghorn chicken	Gallus gallus domesticus	TALEN	OVA	Egg white proteins	[157]
		Gallus gallus domesticus	D10A-Cas9 nickase (Cas9n)	MSTN	Skeletal muscle growth	[159]
	Broiler chicken	Gallus gallus domesticus	CRISPR	G0/G1 (G0S2)	Lipid content/Adipose tissue	[158]
	Japanese quail	Coturnix japonica	CRISPR	MSTN	Skeletal muscle growth	[161]
Pig	Meishan pig	Sus scrofa domesticus	ZFN	MSTN	Skeletal muscle growth	[163,164,165]
	Large White pig	Sus scrofa domesticus	TALEN	MSTN	Skeletal muscle growth	[170]
	Chinese Erhualian pig	Sus scrofa domesticus	CRISPR	MSTN	Skeletal muscle growth	[167]
	-	-	TALEN- SCNT	MSTN	Skeletal muscle growth	[166]
	Chinese experimental mini-pigs	Sus scrofa domesticus	CRISPR- SCNT	Fbxo40	Skeletal muscle growth	[172]
Cattle	Beef cattle		ZFN	MSTN	Skeletal muscle growth	[181]
	Beef cattle	Bos aurus/indicus	TALENs- SCNT	MSTN	Skeletal muscle growth	[135]
	Dairy cattle	Bos taurus	ZFNs- SCNT	BLG	Milk allergen	[178,179]
	Dairy cattle	Bos taurus	CRISPR	BLG	Milk allergen	[180]
	Holstein dairy cattle	Bos taurus	CRISPR	PMEL	Coat color	[187,188]
Sheep	Booroola sheep	Ovis aries	CRISPR	FecB	Reproduction rate	[191]
	Domestic sheep	Ovis aries	CRISPR	HYAL2, O PrP	Reproduction rate	[192]
	Domestic sheep	Ovis aries	CRISPR-Microinjection	MSTN	Skeletal muscle growth	[56,197]
	Merino wool sheep	Ovis aeries	CRISPR	FGF5	Hair growth	[131]
Goat	Arbas cashmere goat	Capra hircus	CRISPR	FAT-1/MSTN	Skeletal muscle growth	[129]
	Dairy goat	Capra aegagrus hircus	TALEN	BLG	Milk allergen	[202]
	Inner Mongolia cashmere goat	Capra spp.	CRISPR	FGF5	Hair growth and development	[208]
	Shaanbei white Cashmere goats	Capra spp.	CRISPR–SCNT	EDAR	Hair growth and development	[208,209]

* Abbreviations: FecB: Booroola fecundity gene; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; dnd: dead-end gene; EDAR: Ectodysplasin receptor-A; eGFP: Enhanced Green Fluorescent Protein gene; FAT-1: FAT atypical cadherin 1; FGF5: Fibroblast growth factor 5 gene; G0/G1 (G0S2): Changeover gene 2; HYAL2: Hyaluronidase 2 gene; LH: Luteinizing hormone gene; LPR: Leptin receptor gene; OVA: Ovalbumin gene; OPrP: Ovine prion Protein; PMEL: Pre-melanosome gene; SCNT: Somatic-cell nuclear transfer; TALEN: Transcription Activator-Like Effector Nuclease; ZFN: Zinc-Finger Endonuclease.

3.1.2. Breeding of Disease-Resistance Animals

One potential application of GE in animals is to produce animals with increased disease resistance [212]. A disease-resistant animal model formed through GE, which is raised in a limited space and susceptible to the spread of disease, can decrease time and economic loss by decreasing dependence on antibiotics. It also can reduce the pain caused by disease and improve animal welfare.

Previously, the two major strategies for disease control were vaccination and antibiotic feeding [213]. However, prolonged use of antibiotics pollutes the environment and expands resistance to harmful microorganisms [213]. Moreover, in traditional breeding methods, it is not easy to choose complex traits that are hard to detect and measure, such as disease-resistance traits. Getting traits such as robust health, elevated muscle mass, and high fecundity through genetics, rather than using antibiotics, growth hormones, or other drugs, is usually favored by both breeders and consumers. For now, the high disease resistance practiced by large-scale animal breeding may cause some production traits [214].

Due to this, researchers have directed their efforts towards modifying animal genomes to improve their disease resistance and general health, assist animal welfare and reproductive efficacy, and develop novel biomedical models to better understand the etiology of diseases [215].

The following sections summarize some of the GE research that has been done to increase resistance to diseases according to species in livestock (Table 4).

Breeding in Fish

One major challenge facing the aquaculture industry is the high mortality rate among fish populations in fish farms [216]. Infectious diseases were the principal cause of mortality, with the most common viral diseases being pancreas disease, gill disease, and heart and skeletal muscle inflammation [216]. A potential strategy for solving problems is to improve fish robustness by adjusting gene control mechanisms implicated in the immune system function [138].

A targeted disturbance of the Toll-like receptor (TLR22) gene was completed in farmed Indian major carp Rohu (Labeo rohita) that can be employed as a model system to study the role of TLR22 against parasites such as fish lice [217] as well as bacteria and pathogenic dsRNA viruses. As an efficient strategy to control the hemorrhagic disease caused by grass carp reovirus (GCRV), CRISPR/Cas9 was used to KO the grass carp (Ctenopharyngodon idellus) Junctional adhesion molecule-A (JAM-A) gene [218]. Recently, the CRISPR-Cas technology has been applied to mutate miRNA binding sites in the three prime untranslated region (3′ UTR) of immune-related genes [138]. Consequently, this prevents miRNA binding and blocks miRNA-induced mRNA breakdown or translational inhibition. Eventually, the mRNA half-life was increased, leading to a notable increase in protein levels that strengthened the immune response.

Breeding in Poultry

Poultry is incredibly susceptible to viruses, such as Mark’s disease virus (MDV), the highly pathogenic avian influenza virus (AIV) [13,124], and the Avian leukosis virus subgroup J (ALV-J) [91,219]. The Chicken Na + /H + exchanger type I (chNHE1)-KO homozygous mutant chicken was successfully engineered using PGCs and CRISPR/Cas9 technologies [219]). This transgenic chicken, which was created by knocking out the retrovirus receptor Tva gene using CRISPR/Cas9, has shown resistance to ALV subgroups A and K, enabling it to combat ALV infection [219].

CRISPR/Cas9 system was utilized to change the recognized residues within the chANP32A gene to diminish AIV replication of the AIV [220]. Also, the CRISPR–Cas9 technique was used to remove Trp38 encoded by chNHE1 W38 of the ALV-J receptor, acquiring the bred chicken resistance against the retroviral infection [219]. In this regard, for the first time, developing a CRISPR–Cas9 vector that stopped the expression of ALV-J in vivo was successful using coinfected MDV. Following this, breeding with GE quickly grows new livestock forms with disease-resistance traits by deleting disease predisposition genes and pathogens receptor genes or introducing disease-resistance genes [13]. Additionally, scientists can test new livestock genetic traits that affect disease resistance and do not exist from natural genetic resources. Recently, chickens resistant to ALV-J were produced by deleting one amino acid, W38, inside the ALV-J receptor NHE1 using CRISPR/Cas9 GE. This resistance phenotype was confirmed in vitro and in vivo [219].

Breeding in Pigs

Swine are one of the primary livestock resources and are considered an ideal animal model for biomedical research and xenotransplantation due to their physiological and genetic similarities to humans [221]. The Porcine Reproductive and Respiratory Syndrome (PRRS) infection is estimated to cost pig farmers about six million dollars daily in North America and Europe [222]. Due to the genetic diversity of the virus, the vaccine has limited efficiency on PRRS [223], and therefore, attention was directed to the cellular receptors involved in the PRRSV entrance into the target cell. Cluster of Differentiation (CD163) is a transmembrane protein on the surface of macrophages acting as a receptor for PRRSV [224]. CRISPR/CAS9 was used to produce a KO mutation in the exon 7 of the CD163 gene [225].

Mutant pigs showed strong resistance to the PRRS virus. Following this, several research groups have successfully produced various types of CD163-KO pigs using CRISPR/Cas9 tools [222,226,227,228,229]. Confrontation with either the NVSL 97-7895 PRRSV virulent virus isolates [225] or the highly pathogenic PRRSV (HP-PRRSV) strain [229] revealed that CD163-KO pigs are resistant to viral disease shown by the nonexistence of viremia, antibody response, high fever or any other PRRS-associated clinical symptoms. Furthermore, neither PRRSV nor its antibodies were found in pig serum. For the first time, gene-edited breeding for livestock disease resistance has solved an obstacle neither selection programs nor vaccines can solve entirely.

One GE design in the specific deletion of the Scavenger Receptor Cysteine-Rich domain 5 (SRCR5) of the CD163 gene while keeping other CD163 domains unharmed. Macrophages from these pigs resisted both PRRSV genotypes [226,227] but not Porcine Epidemic Diarrhea Virus (PEDV) infection after virus challenge tests [227,228]. Furthermore, CRISPR/Cas9 and SCNT were utilized to produce pigs with the concurrent KO of CD163 and Porcine Aminopeptidase N (APN) genes [207]. Further analysis revealed that the double-KO (DKO) pigs were utterly resistant to genotype 2 PRRSV and transmissible gastroenteritis virus (TGEV) infection simultaneously. These findings confirmed that DKO CD163 and pAPN pigs were produced [230]. This study reported that the pigs with DKO mutation had no difference in meat production and reproductive performance relative to wild-type pigs. Lung fibroblast-like cells originating from these animals maintained a prominent level of Porcine Delta Coronavirus (PDCoV) infection, suggesting that APN is a dispensable receptor for PDCoV [231]. These CD163 null mutations resist several PRRSV isolates [222,229].

Additionally, many GM pigs created using CRISPR/Cas9 are appropriate donors for disease models and xenotransplantation [107,229,232]. Transgenic pigs resistant to the classical swine fever virus (CSFV) were bred using CRISPR–Cas9 [233]. These researchers observed that growing pigs, pregnant sows, and fetuses were resistant to PRRSV infection. CD163 KO pigs were also bred, and the replication of CSFV was confined while disease resistance was stably transferred to offspring. The African swine fever virus (ASFV) has a higher mortality rate than ordinary swine fever, reaching 100% in some cases. CRISPR–Cas9 was used to target ASFV phosphoprotein p30 to decrease the number of viruses by inhibiting viral replication [234]. In 2020, the CD163 and pAPN DKO pigs were assembled [207], turning off the virus receptor CD163 and pAPN proteins and confirming more excellent disease resistance. Other infection challenge trials have indicated that these DKO pigs showed reduced susceptibility to PDCoV, providing in vivo evidence that pAPN could serve as one of the receptors for PDCoV. This procedure blocked PRRSV and TGEV infections and conserved reproductive capacity and meat quality [235].

Finally, it should be mentioned that knocking out of another PRRSV receptor gene, CD169, was attempted, but no significant differences in the viral challenge were reported between edited and wild-type pigs [222]. This negative finding suggests that this gene may not necessarily affect the occurrence of PRRS. Commentators have stressed that, at present, GE is most useful for traits affected by one major gene (monogenic) [236], for instance, resistance to PRRS. Monogenic resistance to infections in pigs is rare [237], and conventional breeding is likely not to be used for traits affected by multiple genes in livestock [137]. The Royal Society for the Protection of Cruelty to Animals debated that genome-edited disease resistance can be short-lived if diseases develop to overcome changes in single genes [238].

Breeding in Cattle

Mycobacterium bovis (Mbv) infection can cause large debts to the livestock business and poses zoonotic dangers because humans mainly acquire Mby infection through infected food or drinks, unpasteurized dairy foods, or exposure to infected animals [103].

In cattle, bovine CD18 is a gene that codes for the leukotoxin receptor found on the leucocyte surface. The use of ZFNs to insert a single amino acid substitution (glutamine [Q] to glycine [G]) at amino acid position five in both alleles resulted in the production of transgenic cattle resistant to the cytotoxicity of leukotoxin secreted by Mannheimia (Pasteurella) haemolytica, the bacterium responsible for the respiratory disease Pasteurellosis [239] (Table 4). This study confirmed the feasibility of developing gene-modified cattle resistant to M. haemolytica-caused pneumonia.

The TALE nickase-mediated SP110 gene KI was utilized to produce Tuberculosis (TB)-resistant cattle, and the gene-edited cows were capable of inhibiting the growth and reproduction of Mby [232]. Also in bovine, Gao et al. [240] were the first researchers to use Cas9 nickase (Cas9n)-mediated homologous recombination to integrate naturally resistant-associated macrophage protein-1 (NRAMP1) into the bovine genome, promoting their resistance to Mbv infection. Combined with SCNT, gene-edited cattle with significantly enhanced TB resistance were produced. Gene-edited TB-resistance cattle were generated using the CRISPR/Cas 9 with NRAMP gene inclusion [241] (Table 4).

The CRISPR–Cas9 strategy was used to develop a CD46 gene KO cell line reliant on the highly contagious bovine viral diarrhea virus (BVDV) attack [242]. This virus can cause severe intestinal and respiratory diseases in cattle. The virus also causes abortion in pregnant cows, and certain infected calves that acquire it at birth can transmit the infection for the rest of their lives. These experiments enabled researchers to clarify the mechanism underlying CD46’s role in plague viruses’ replication cycles. The virus binds to a cellular receptor (CD46), causing infection in cows. In vivo, CRISPR-mediated homology-directed repair and SCNT were used to generate a mutation in the BVDV binding domain of bovine CD46 [243]. The outcome was a GE CD46 receptor in the virus unable to bind and a live calf with drastically reduced susceptibility to the infection. The first CD46 gene-edited calf was produced.

Table 4:

Examples of transgenic farmed animals produced using gene editing strategies for disease-resistance.

Animal	Common Name	Scientific Name	Editing Tool	Edited Gene Abbreviation *	Trait Targeted	Reference
Fish	Grass carp	Ctenopharyngodon idellus	RISPR	JAM-A	GCRV hemorrhagic disease infection	[218]
Poultry	Broiler chicken	Gallus gallus domesticus	CRISPR-MIPGCs	chNHE1	ALV infection	[219]
Pig	-	Sus domesticus	CRISPR	CD163	Genotype 1 PRRS infection	[225]
	Chinese pig	Sus domesticus	CRISPR-SCNT	CD163	Genotype 1 PRRS infection	[227]
	Wild pig	Sus scrofa	CRISPR- SCNT	CD163/APN	Genotype 2 PRRSV/TGEV infections	[207,230]
Cattle	Cow	Bos taurus	ZFN	CD18	Pasteurellosis resistance	[239]
	Holstein–Friesian dairy cow	Bos taurus	TALE	SP110	Tuberculosis resistance	[232]
	Holstein–Friesian dairy cow	Bos taurus	Single Cas9n- SCNT	NRAMP	Tuberculosis resistance	[240,241]
	Mardin-Darby cow	Bos taurus	CRISPR- SCNT	CD46	BVDV resistance	[242]
	Chinese dairy cow	Bos taurus	ZFN-SCNT	CSN2	Mastitis resistance	[244]
	Holstein–Friesian dairy cow	Bos taurus	TALEN	SP110/MAT1A/ SFTPA1	Tuberculosis resistance	[245]
	Holstein–Friesian diary cow	Bos taurus	CRISPR	NRAMP1 /FSCN1-ACTB (F-A)	Tuberculosis resistance	[245]
Sheep	Domestic sheep	Ovis aries	CRISPR–SCNT	IFNA	ZIKV resistance	[233]

* Abbreviations: ACTB: Beta-actin gene; ALV: Avian leukosis virus; APN: Aminopeptidase N gene; APOL1: Apolipoprotein L1 gene; BVDV: Bovine viral diarrhea virus; Cas9n: Cas9 nickase; CSN2: Casein beta gene; CD: Cluster of Differentiation gene; CSFV: Classical swine fever virus; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; FSCN1: Fascin actin-bundling protein 1 gene; GCRV: Grass carp reovirus; chNHE1: Chicken Na + /H + exchanger type I gene; IFNA: Interferon Alpha gene; JAM-A: Junctional adhesion molecule-A gene; MAT1A: Methionine Adenosyl transferase 1a gene; MI: Microinjection; NRAMP1: Naturally resistant-associated macrophage protein-1; (PRRS: Porcine Reproductive and Respiratory Syndrome; PGCs: Primordial Germ Cells; SFTPA1: Pulmonary surfactant protein A1; SCNT: Somatic-cell nuclear transfer; SP110: SP110 nuclear body protein gene; TGEV: Transmissible gastroenteritis virus; ZIKV: Zika virus.

In dairy cows, Mastitis is a severe disease caused by Bovine paratuberculosis, resulting in considerable economic losses to the dairy industry. Using ZFNs-mediated gene recombination approach, the human lysozyme gene and lysostaphin gene were precisely inserted into the endogenous Casein beta (CSN2) gene locus of the cow [244] (Table 4). Then, transgenic cows were produced by SCNT. The gene-edited cows were capable of secreting lysostaphin or human lysozyme, and consequently killing Staphylococcus aureus in their milk [244]. Furthermore, the ZFN GE method was used to direct the human lysozyme gene to the beta-casein locus, creating a transgenic mastitis resistance cow [183].

Using TALEN, the mouse SP110 gene was introduced into the Methionine Adenosyltransferase 1a (MAT1A) and Pulmonary surfactant protein A1 (SFTPA1) sites of the Holstein-Friesian dairy cow genome [232]. Integration of the NRAMP1 gene into the Fascin actin-bundling protein 1- Beta-actin (FSCN1-ACTB) locus and the bovine homology of the mouse Rosa26 locus using the CRISPR/Cas9 system was a significant step. This genetic modification conferred high resistance in the NRAMP1 transgenic cows against tuberculosis [232], underscoring the potential of genetic research in enhancing disease resistance in livestock. The transgenic cows showed elevated resistance to Mbv infection. In vitro, CRISPR/Cas9-mediated NO of the CD46 gene in Madin–Darby bovine kidney (MDBK) cells led to a significant decline in BVDV susceptibility [242].

Breeding in Sheep and Goats

As sheep’s body size and functional structure are comparable to those of humans, CRISPR–Cas9 tools have been used in sheep to offer an effective model for studying disease resistance breeding for several diseases. Therefore, the CRISPR–Cas9 system provides a promising therapeutic model for investigating pathogenesis and improving animal health. The thymosin β4 (Tβ4) gene was implanted into the chemokine receptor type 5 (CCR5) site in goats, enabling the establishment of a goat knock-in model [195].

African researchers are now working to use the CRISPR/Cas9 system to KI the Apolipoprotein L1 (ApoL 1) gene, which confers resistance to trypanosomiasis in primates [245], into an Indigenous goat breed [246]. If successful, this GE scheme could also be used to oppose the destructive disease of trypanosomiasis in Indigenous cattle breeds. The phenotypes of the liver and gallbladder diseases of newborn Cystic Fibrosis Transmembrane conductance regulator (CFTR)-/- sheep agreed with those of humans. In sheep, CRISPR–Cas9 was applied to induce one point mutation in the tissue non-specific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) [247]. The resulting engineered lambs exhibited a rare human bone disease, hypophosphatasia, thus, providing an efficient animal model for investigating this rare metabolic bone disease. Also, in lambs, the possibility of using CRISPR–Cas9 for engineering antiviral animals has been established [248]. The hyaluronidase 2 (HYAL2) gene involved in lung adenocarcinoma syndrome was KO. It was for the first time that removing the sheep otoferlin (OOF) gene is an efficient model for treating deafness [248]. Another report pointed out that the CRISPR/Cas9 system adjusts HYAL2 and PrP genes to produce disease-resistant lambs [192].

The CRISPR–Cas9 and SCNT technologies were combined to breed Interferon Alpha (IFNA) gene KO sheep [233]. This engineered sheep offered a large-scale animal model for fetal resistance to Zika virus (ZIKV) infection (Table 4). Thereafter, using the same technology, the human cystic fibrosis (CF) sheep model with CRISPR–Cas9–mediated CFTR deficiency was constructed [249]. The SCNT program targeted KO PRP in goat fibroblast donor cells to generate SCNT-mediated anti-PRP goats. The CRISPR–Cas9 produced the NUP155 gene KO donor cells in goat fibroblasts [249]. These experiments developed a NUP155 gene KO goat model for investigating heart disease. Pancreas/duodenum homeobox protein 1 (PDX1) fetuses were composed [250], permitting gene-edited sheep to serve as host organs for xenotransplantation. Furthermore, interspecies blastocyst-paired technology, combining embryonic gene editing and pluripotent stem cells (PSCs), has enabled xenotransplantation of human organs by using large animals as hosts [104].

These studies confirm that the CRISPR/Cas9 system can significantly improve modern livestock breeding systems. The DNA BE can produce new varieties without limitation to individuals or species. Single nucleotide variants (SNVs) are the critical genetic basis for trait alteration in livestock and the top cause of 2/3 of human diseases [251]. Therefore, developing a new BE system in GE technology based on the CRISPR/Cas system is very important and urgent [91].

4. Conclusions and Future Perspectives

Abbreviations

Author Contributions

Conflicts of Interest

Acknowledgments

Funding

References

[1] , "Volume II: Demographic Profiles (ST/ESA/SER.A/400). Report," in World Population Prospects: The 2017 Revision, , Eds. New York, USA: United Nations, Department of Economic and Social Affairs, Population Division, 2017, .

[2] , "," in The State of World Fisheries and Aquaculture: Sustainability in Action, , Eds. Rome, Italy: FAO, 2020, . Available online: https://doi.org/10.4060/ca9229en.

[3] D.-H. Kwon, G.-M. Gim, S.-Y. Yum, G. Jang, "Current status and future of gene engineering in livestock" BMB Rep., vol. 57, pp. 50-59, 2024. [Crossref]

[4] R. Jasrotia, M. Dhar, S. Langer, "Climate change impacts on animal production," in Global Agricultural Production: Resilience to Climate Change, M. Ahmed, Eds. London, UK: Springer, 2023, pp. 311-333.

[5] G.M. Gim, G. Jang, "Outlook on genome editing application to cattle" J. Vet. Sci., vol. 25, p. e10, 2024. [Crossref]

[6] M. Chui, M. Evers, J. Manyika, A. Zheng, T. Nisbet, "The bio revolution: Innovations transforming economies, societies, and our lives," in Augmented Education in the Global Age: Artificial Intelligence and the Future of Learning and Work, D. Araya and P. Marber, Eds. New York, NY, USA: Routledge, 2023, pp. 48-74.

[7] J.H. Han, J.S. Yu, D.H. Kim, H.W. Choi, "The characteristics of bovine satellite cells with highly scored genomic estimated breeding value" J. Anim. Reprod. Biotechnol., vol. 38, pp. 177-187, 2023. [Crossref]

[8] S. Roy, V. Kumar, B.K. Behera, J. Parhi, S. Mohapatra, T. Chakraborty, et al., "CRISPR/Cas genome editing—Can it become a game changer in future fisheries sector?" Front. Mar. Sci., vol. 9, p. 924475, 2022. [Crossref]

[9] B.P. Telugu, K.E. Park, C.H. Park, "Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications" Mamm. Genome, vol. 28, pp. 338-347, 2017. [Crossref]

[10] F. Gao, N. Hou, X. Du, Y. Wang, J. Zhao, S. Wu, "Molecular breeding of farm animals through gene editing" Natl. Sci. Open, vol. 2, p. 20220066, 2023. [Crossref]

[11] S.G. Lillico, C. Proudfoot, T.J. King, W. Tan, L. Zhang, R. Mardjuki, et al., "Mammalian interspecies substitution of immune modulatory alleles by genome editing" Sci. Rep., vol. 6, 2016. [Crossref] [PubMed]

[12] I.V. Perisse, Z. Fan, G.N. Singina, K.L. White, I.A. Polejaeva, "Improvements in gene editing technology boost its applications in livestock" Front. Genet., vol. 11, 2021. [Crossref]

[13] Z. Liu, T. Wu, G. Xiang, H. Wang, B. Wang, Z. Feng, et al., "Enhancing animal disease resistance, production efficiency, and welfare through precise genome editing" Int. J. Mol. Sci., vol. 23, 2022. [Crossref]

[14] M. Yousuf, A. Yusuf, I. Mohammed, "Review on current animal breeding and genetic technologies to increase production and productivity of cattle" Glob. J. Anim. Sci. Res., vol. 12, pp. 19-26, 2024. Available at: http://www.gjasr.com/index.php/GJASR/article/view/191.

[15] K. Kumar, G. Gambhir, A. Dass, A.K. Tripathi, A. Singh, A.K. Jha, et al., "Genetically modified crops: Current status and future prospects" Planta, vol. 251, p. 91, 2020. [Crossref] [PubMed]

[16] V. Kilders, A. Ali, "Understanding the influence of end-users on the acceptance of gene edited foods and sensitivity to information" Food Qual. Prefer., vol. 120, p. 105238, 2024. [Crossref]

[17] C. Denning, H. Priddle, "New frontiers in gene targeting and cloning: Success, application and challenges in domestic animals and human embryonic stem cells" Reproduction, vol. 126, pp. 1-11, 2003. [Crossref]

[18] E. Melo, A. Canavessi, M. Franco, R. Rumpf, "Animal trangenesis: State of the art and applications" J. Appl. Genet., vol. 48, pp. 47-61, 2007. [Crossref] [PubMed]

[19] K. Lee, K. Uh, K. Farrell, "Current progress of genome editing in livestock" Theriogenology, vol. 150, pp. 229-235, 2020. [Crossref]

[20] I. Wilmut, A.E. Schnieke, J. McWhir, A.J. Kind, K.H. Campbell, "Viable offspring derived from fetal and adult mammalian cells" Nature, vol. 385, pp. 810-813, 1997. [Crossref]

[21] L. Lai, D. Kolber-Simonds, K.W. Park, H.T. Cheong, J.L. Greenstein, G.S. Im, et al., "Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning" Science, vol. 295, pp. 1089-1092, 2002. [Crossref]

[22] S.Y. Yum, K.Y. Youn, W.J. Choi, G. Jang, "Development of genome engineering technologies in cattle: From random to specific" J. Anim. Sci. Biotechnol., vol. 9, 2018. [Crossref]

[23] A.L. Van Eenennaam, F.D. Silva, J.F. Trott, D. Zilberman, "Genetic engineering of livestock: The opportunity cost of regulatory delay" Annu. Rev. Anim. Biosci., vol. 9, pp. 453-478, 2021. [Crossref]

[24] H.B. Kaya, "Base editing and prime editing," in A Roadmap for Plant Genome Editing, A. Ricroch and D. Eriksson and D. Miladinović and J. Sweet and K. Van Laere and E. Woźniak-Gientka, Eds. Cham, Switzerland: Springer Nature, 2024, pp. 17-39.

[25] M. Qaim, "Role of new plant breeding technologies for food security and sustainable agricultural development" Appl. Econ. Perspect. Policy, vol. 42, pp. 129-150, 2020. [Crossref]

[26] A.M. Khalil, "Genome editing in plants via transcription activator-like effector nucleases (TALENs)," in Plant Genome Editing Development and Technologies, K.E.M. Salem and J.M. Al-Khayri and S.M. Jain, Eds. Cham, Switzerland: Springer, 2025, . (In Press)

[27] J.M. Hickey, T. Chiurugwi, I. Mackay, W. Powell, "Genomic prediction unifies animal and plant breeding programs to form platforms for biological discovery" Nat. Genet., vol. 49, pp. 1297-1303, 2017. [Crossref] [PubMed]

[28] E.M. Hallerman, J.P. Bredlau, L.S.A. Camargo, M.L.Z. Dagli, M. Karembu, G. Ngure, et al., "Towards progressive regulatory approaches for agricultural applications of animal biotechnology" Transgenic Res., vol. 31, pp. 167-199, 2022. [Crossref]

[29] M. Platani, O. Sokefun, E. Bassil, Y. Apidianakis, "Genetic engineering and genome editing in plants, animals, and humans: Facts and myths" Gene, vol. 856, p. 147141, 2022. [Crossref] [PubMed]

[30] D. Lim, I. Choi, "Global trends in regulatory frameworks for animal genome editing in agriculture" J. Anim. Reprod. Biotechnol., vol. 38, pp. 247-253, 2023. [Crossref]

[31] K. Kawall, J. Cotter, C. Then, "Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture" Environ. Sci. Eur., vol. 32, p. 106, 2020. [Crossref]

[32] R. Jaenisch, "Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus" Proc. Natl. Acad. Sci. USA, vol. 73, pp. 1260-1264, 1976. [Crossref] [PubMed]

[33] C. Rexroad, J. Vallet, L.K. Matukumalli, J. Reecy, D. Bickhart, H. Blackburn, et al., "Genome to phenome: Improving animal health, production, and well-being—A New USDA blueprint for animal genome research 2018–2027" Front. Genet., vol. 10, 2019. [Crossref]

[34] A.M. Khalil, "Cell delivery strategies: Review of the recent developments, challenges, and opportunities" Int. J. Appl. Sci. Res. Rev., vol. 9, p. 60, 2022. Available at: https://www.primescholars.com/articles/cell-delivery-strategies-review-of-the-recent-developments-challenges-and-opportunities-111621.html.

[35] J. Popova, V. Bets, E. Kozhevnikova, "Perspectives in genome-editing techniques for livestock" Animals, vol. 13, 2023. [Crossref]

[36] Q.A. Le, F. Tanihara, M. Wittayarat, Z. Namula, Y. Sato, Q. Lin, et al., "Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages" BMC Res. Notes, vol. 14, 2021. [Crossref]

[37] X. Yao, M. Zhang, X. Wang, W. Ying, X. Hu, P. Dai, et al., "Tild-CRISPR allows for efficient and precise gene knock-in in mouse and human cells" Dev. Cell, vol. 45, pp. 526-536.e5, 2018. [Crossref] [PubMed]

[38] H. Chen, Y. Li, T.O. Tollefsbol, "Cell senescence culturing methods" Methods Mol. Biol., vol. 1048, pp. 1-10, 2013. [Crossref] [PubMed]

[39] W.H. Eyestone, "Production and breeding of transgenic cattle using in vitro embryo production technology" Theriogenology, vol. 51, pp. 509-517, 1999. [Crossref]

[40] R.J. Wall, "Transgenic livestock: Progress and prospects for the future" Theriogenology, vol. 45, pp. 57-68, 1996. [Crossref]

[41] P. Krimpenfort, A. Rademakers, W. Eyestone, A. van der Schans, S. van den Broek, P. Kooiman, et al., "Generation of transgenic dairy cattle using ‘in vitro’ embryo production" Nat. Biotechnol., vol. 9, pp. 844-847, 1991. [Crossref] [PubMed]

[42] R.E. Hammer, V.G. Pursel, C.E. Rexroad, R.J. Wall, D.J. Bolt, K.M. Ebert, et al., "Production of transgenic rabbits, sheep, and pigs by microinjection" Nature, vol. 315, pp. 680-683, 1985. [Crossref]

[43] L. Jiang, J. Liu, D. Sun, P. Ma, X. Ding, Y. Yu, et al., "Genome-wide association studies for milk production traits in Chinese Holstein population" PLoS ONE, vol. 5, 2010. [Crossref]

[44] S. Gottlieb, M.B. Wheeler, "," in Genetically engineered animals and public health: Compelling benefits for health care, nutrition, the environment, and animal welfare, , Eds. Washington, DC, USA: American Enterprise Institute, 2011, pp. 22-30. Available at: https://experts.illinois.edu/en/publications/genetically-engineered-animals-and-public-health-compelling-benef.

[45] R. Li, Q. Meng, J. Qi, L. Hu, J. Huang, Y. Zhang, et al., "Microinjection-based CRISPR/Cas9 mutagenesis in the decapoda crustaceans Neocaridina heteropoda and Eriocheir sinensis" J. Exp. Biol., vol. 225, 2022. [Crossref] [PubMed]

[46] E. Ayana, G. Fentahun, A. Negash, F. Mitku, M. Zemene, F. Zeru, "Review on applications of genetic engineering and cloning in farm animals" J. Dairy. Vet. Sci., vol. 4, p. JDVS.MS-ID.555629, 2017. [Crossref]

[47] Z. Yang, Y. Yu, Y.X. Tay, G.H. Yue, "Genome editing and its applications in genetic improvement in aquaculture" Rev. Aquac., vol. 14, pp. 178-191, 2022. [Crossref]

[48] M. Asaye, H. Biyazen, M. Girma, "Genetic engineering in animal production: Applications and prospects" Biochem. Biotechnol. Res., vol. 2, pp. 12-22, 2014. Available at: https://www.netjournals.org/z_BBR_14_013.html.

[49] R.L. Gratacap, T. Regan, C.E. Dehler, S.A.M. Martin, P. Boudinot, B. Collet, et al., "Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system" BMC Biotechnol., vol. 20, 2020. [Crossref]

[50] Y. Kindinew, G. Abaynew, F. Haben, "Genetic engineering application in animal breeding—Review" Biomed. J. Sci. Tech. Res., vol. 32, pp. 25180-25188, 2020. [Crossref]

[51] V.G. Pursel, C.E. Rexroad, "Status of research with transgenic farm-animals" J. Anim. Sci., vol. 71, pp. 10-19, 1993. [Crossref]

[52] S.L. Hennig, J.R. Owen, J.C. Lin, A.E. Young, P.J. Ross, A.L. Van Eenennaam, et al., "Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos" Sci. Rep., vol. 10, 2020. [Crossref] [PubMed]

[53] M. Gajecka, "Unrevealed mosaicism in the next-generation sequencing era" Mol. Genet. Genomics, vol. 291, pp. 513-530, 2016. [Crossref]

[54] Z. Mclean, B. Oback, G. Laible, "Embryo-mediated genome editing for accelerated genetic improvement of livestock" Front. Agr. Sci. Eng., vol. 7, pp. 148-160, 2020. [Crossref]

[55] T. Hai, F. Teng, R. Guo, W. Li, Q. Zhou, "One-step generation of knockout pigs by zygote injection of CRISPR/Cas system" Cell Res., vol. 24, pp. 72-375, 2014. [Crossref]

[56] M. Crispo, A. Mulet, L. Tesson, N. Barrera, F. Cuadro, P. Dos Santos-Neto, et al., "Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes" PLoS ONE, vol. 10, 2015. [Crossref]

[57] B. Petersen, A. Frenzel, A. Lucas-Hahn, D. Herrmann, P. Hassel, S. Klein, et al., "Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes" Xenotransplantation, vol. 23, pp. 338-346, 2016. [Crossref]

[58] S. Chen, S. Sun, D. Moonen, C. Lee, A.Y. Lee, D.V. Schaffer, et al., "CRISPR-READI: Efficient generation of knockin mice by CRISPR RNP electroporation and AAV donor infection" Cell Rep., vol. 27, pp. 3780-3789.e3784, 2019. [Crossref]

[59] T.F. Bishop, A.L. Van Eenennaam, "Genome editing approaches to augment livestock breeding programs" J. Exp. Biol., vol. 223, p. jeb207159, 2020. [Crossref]

[60] J.C. Lin, A.L. Van Eenennaam, "Electroporation-mediated genome editing of livestock zygotes" Front. Genet., vol. 12, 2021. [Crossref] [PubMed]

[61] M. Kaulich, Y.J. Lee, P. Lönn, A.D. Springer, B.R. Meade, S.F. Dowdy, "Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi" Nucleic Acids Res., vol. 43, p. e45, 2015. [Crossref]

[62] H. Yin, K. Kauffman, D. Anderson, "Delivery technologies for genome editing" Nat. Rev. Drug Discov., vol. 16, pp. 387-399, 2017. [Crossref] [PubMed]

[63] Y. Yoon, D. Wang, P.W.L. Tai, J. Riley, G. Gao, J.A. Rivera-Pérez, "Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses" Nat. Commun., vol. 9, p. 412, 2018. [Crossref] [PubMed]

[64] G.R. McFarlane, H.A. Salvesen, A. Sternberg, S.G. Lillico, "On-farm livestock genome editing using cutting edge reproductive technologies" Front. Sustain. Food Syst., vol. 3, 2019. [Crossref]

[65] T. Kaneko, T. Mashimo, "Simple genome editing of rodent intact embryos by electroporation" PLOS ONE, vol. 10, 2015. [Crossref]

[66] T.A. Larkina, A. Krutikova, G. Peglivanyan, Y.S.O. Shcherbakov, O. Barkova, "Development of optimal technological approaches for obtaining PGCs in Pushkin breed chickens for further transformation by the CRISPR/Cas9 system" FASEB J., vol. 35, 2021. [Crossref]

[67] Y. Dai, T.D. Vaught, J. Boone, S.H. Chen, C.J. Phelps, S. Ball, et al., "Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs" Nat. Biotechnol., vol. 20, pp. 251-255, 2002. [Crossref]

[68] V. Zakhartchenko, S. Mueller, R. Alberio, W. Schernthaner, M. Stojkovic, H. Wenigerkind, et al., "Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts" Mol. Reprod. Dev., vol. 60, pp. 362-369, 2001. [Crossref] [PubMed]

[69] R.C. Reggio, A.N. James, H.L. Green, W.G. Gavin, E. Behboodi, Y. Echelard, et al., "Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: Oocytes derived from both follicle-stimulating hormone-stimulated and nonstimulated abattoir-derived ovaries, Biol" Reprod., vol. 65, pp. 1528-1533, 2001. [Crossref] [PubMed]

[70] M. Skrzyszowska, M. Samiec, "Generating cloned goats by somatic cell nuclear transfer—Molecular determinants and application to transgenics and biomedicine" Int. J. Mol. Sci., vol. 22, 2021. [Crossref]

[71] X. Tian, C. Kubota, B. Enright, X. Yang, "Cloning animals by somatic cell nuclear transfer—Biological factors" Reprod. Biol. Endocrinol., vol. 1, 2003. [Crossref]

[72] J.B. Cibelli, S.L. Stice, P.J. Golueke, J.J. Kane, J. Jerry, C. Blackwell, et al., "Cloned transgenic calves produced from nonquiescent fetal fibroblasts" Science, vol. 280, pp. 1256-1258, 1998. [Crossref] [PubMed]

[73] A. Baguisi, E. Behboodi, D.T. Melican, J.S. Pollock, M.M. Destrempes, C. Cammuso, et al., "Production of goats by somatic cell nuclear transfer" Nat. Biotechnol., vol. 17, pp. 456-461, 1999. [Crossref]

[74] K.W. Park, H.T. Cheong, L. Lai, G.S. Im, B. Kuhholzer, A. Bonk, et al., "Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein" Anim. Biotechnol., vol. 12, pp. 173-181, 2001. [Crossref]

[75] I. Polejaeva, K. Campbell, "New advances in somatic cell nuclear transfer: Application in transgenesis" Theriogenology, vol. 53, pp. 117-126, 2000. [Crossref]

[76] A.E. Schnieke, A.J. Kind, W.A. Ritchie, K. Mycock, A.R. Scott, M. Ritchie, et al., "Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts" Science, vol. 278, pp. 2130-2133, 1997. [Crossref]

[77] L. Lai, J.X. Kang, R. Li, J. Wang, W.T. Witt, H.Y. Yong, et al., "Generation of cloned transgenic pigs rich in omega-3 fatty acids" Nat. Biotechnol., vol. 24, pp. 435-436, 2006. [Crossref]

[78] K.J. McCreath, J. Howcroft, K.H. Campbell, A. Colman, A.E. Schnieke, A.J. Kind, "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells" Nature, vol. 405, pp. 1066-1069, 2000. [Crossref] [PubMed]

[79] M. Panarace, J.I. Agüero, M. Garrote, G. Jauregui, A. Segovia, L. Cané, et al., "How healthy are clones and their progeny: 5 years of field experience" Theriogenology, vol. 67, pp. 142-151, 2007. [Crossref]

[80] M.R. Park, S.K. Cho, S.Y. Lee, Y.J. Choi, J.Y. Park, D.N. Kwon, et al., "A rare and often unrecognized cerebromeningitis and hemodynamic disorder: A major cause of sudden death in somatic cell cloned piglets" Proteomics, vol. 5, pp. 1928-1939, 2005. [Crossref]

[81] T. Liu, H. Dou, X. Xiang, L. Li, Y. Li, L. Lin, et al., "Factors determining the efficiency of porcine somatic cell nuclear transfer: Data analysis with over 200,000 reconstructed embryos" Cell Reprog., vol. 17, pp. 463-471, 2015. [Crossref]

[82] J.R. Hill, "Incidence of abnormal offspring from cloning and other assisted reproductive technologies" Annu. Rev. Anim. Biosci., vol. 2, pp. 307-321, 2014. [Crossref]

[83] P.C. Ronald, M. Kliegman, "CRISPR in Agriculture," in CRISPRpedia, H.R. Henderson and M.L. Hochstrasser and T. Tolpa and B. Cheung and G. Ramit and A.G. Murdock and M. Kliegman and P.C. Ronald, Eds. Berkeley, CA, USA: Innovative Genomics Institute, University of California, Berkeley, 2022, . Available online: https://innovativegenomics.org/crisprpedia/crispr-in-agriculture/.

[84] Z. Ruan, X. Zhao, X. Qin, C. Luo, X. Liu, Y. Deng, et al., "DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes" Reprod. Domest. Anim., vol. 53, pp. 203-212, 2017. [Crossref] [PubMed]

[85] K.M. Whitworth, K. Lee, J.A. Benne, B.P. Beaton, L.D. Spate, S.L. Murphy, et al., "Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro derived oocytes and embryos" Biol. Reprod., vol. 91, 2014. [Crossref] [PubMed]

[86] A. Ogura, K. Inoue, T. Wakayama, "Recent advancements in cloning by somatic cell nuclear transfer" Philos. Trans. R. Soc. B Biol. Sci., vol. 368, 2013. [Crossref]

[87] F.H. Biase, C. Rabel, M. Guillomot, I. Hue, K. Andropolis, C.A. Olmstead, et al., "Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium" Proc. Natl. Acad. Sci. USA, vol. 113, pp. 14492-14501, 2016. [Crossref]

[88] G. Gao, S. Wang, J. Zhang, G. Su, Z. Zheng, C. Bai, et al., "Transcriptome-wide analysis of the SCNT bovine abnormal placenta during mid- to late gestation" Sci. Rep., vol. 9, 2019. [Crossref]

[89] C. Zhou, Y. Wang, J. Zhang, J. Su, Q. An, X. Liu, et al., "H3K27me3 is an epigenetic barrier while KDM6A overexpression improves nuclear reprogramming efficiency" FASEB J., vol. 33, pp. 4638-4652, 2019. [Crossref]

[90] S. Wang, Z. Qu, Q. Huang, J. Zhang, S. Lin, Y. Yang, et al., "Application of gene editing technology in resistance breeding of livestock" Life, vol. 12, 2022. [Crossref] [PubMed]

[91] H.I. Ahmad, N. Bibi, J. Abdul, "The evolution of genome-editing technologies," in OMICs-Based Techniques for Global Food Security, S. Fiaz and C.S. Prakash, Eds. New York, NY, USA: John Wiley and Sons, Ltd., 2024, pp. 171-188.

[92] N. Haruyama, A. Cho, A.B. Kulkarni, "Overview: Engineering transgenic constructs and mice" Curr. Protoc. Cell Biol., vol. 42, pp. 19.10.1-19.10.9, 2009. [Crossref] [PubMed]

[93] F. Bushman, M. Lewinski, A. Ciuffi, S. Barr, J. Leipzig, S. Hannenhalli, et al., "Genome-wide analysis of retroviral DNA integration" Nat. Rev. Microbiol., vol. 3, pp. 848-858, 2005. [Crossref] [PubMed]

[94] A. Fernández, S. Josa, L. Montoliu, "A history of genome editing in mammals" Mamm. Genome, vol. 28, pp. 237-246, 2017. [Crossref]

[95] A.M. Khalil, "The genome editing revolution" J. Genet. Eng. Biotechnol., vol. 18, 2020. [Crossref]

[96] X. Xu, Y. Yuan, B. Feng, W. Deng, "CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement" Food Qual. Saf., vol. 4, pp. 159-166, 2020. [Crossref]

[97] H. Yang, S. Ren, S. Yu, H. Pan, T. Li, S. Ge, et al., "Methods favoring homology directed repair choice in response to CRISPR/Cas9 induced-double strand breaks" Int. J. Mol. Sci., vol. 21, 2020. [Crossref]

[98] J.P. Zhang, X.L. Li, G.H. Li, W. Chen, C. Arakaki, G.D. Botimer, et al., "Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage" Genome Biol., vol. 18, 2017. [Crossref]

[99] K.A. Molla, J. Shih, M.S. Wheatley, Y. Yang, "Predictable NHEJ insertion and assessment of HDR editing strategies in plants" Front. Genome Ed., vol. 4, 2022. [Crossref]

[100] I.C. Kurt, R. Zhou, S. Iyer, S.P. Garcia, B.R. Miller, L.M. Langner, et al., "CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells" Nat. Biotechnol., vol. 39, pp. 41-46, 2021. [Crossref]

[101] A.V. Anzalone, L.W. Koblan, D.R. Liu, "Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors" Nat. Biotechnol., vol. 38, pp. 824-844, 2020. [Crossref]

[102] S. Fiaz, S. Ahmar, S. Saeed, A. Riaz, F. Mora-Poblete, K.-H. Jung, "Evolution and application of genome editing techniques for achieving food and nutritional security" Int. J. Mol. Sci., vol. 22, 2021. [Crossref] [PubMed]

[103] S.H.A. Raza, A.A. Hassanin, S.D. Pant, S. Bing, M.Z. Sitohy, S.A. Abdelnour, et al., "Potentials, prospects and applications of genome editing technologies in livestock production" Saudi J. Biol. Sci., vol. 29, pp. 1928-1935, 2022. [Crossref]

[104] Z. Lu, L. Zhang, Q. Mu, J. Liu, Y. Chen, H. Wang, et al., "Progress in research and prospects for application of precision gene-editing technology based on CRISPR–Cas9 in the genetic improvement of sheep and goats" Agriculture, vol. 14, 2024. [Crossref]

[105] K. Fischer, B. Rieblinger, R. Hein, R. Sfriso, J. Zuber, A. Fischer, et al., "Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2" Xenotransplantation, vol. 27, p. e12560, 2020. [Crossref] [PubMed]

[106] L. Cong, F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, et al., "Multiplex genome engineering using CRISPR/Cas systems" Science, vol. 339, pp. 819-823, 2013. [Crossref]

[107] D. Niu, H.J. Wei, L. Lin, H. George, T. Wang, I.H. Lee, et al., "Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9" Science, vol. 357, pp. 1303-1307, 2017. [Crossref]

[108] S. Navarro-Serna, M. Vilarino, I. Park, J. Gadea, P.J. Ross, "Livestock gene editing by one-step embryo manipulation" J. Equ. Vet. Sci., vol. 89, p. 103025, 2020. [Crossref]

[109] P. Singh, S.A. Ali, "Impact of CRISPR-Cas9-based genome engineering in farm animals" Vet. Sci., vol. 8, 2021. [Crossref]

[110] D. Miao, M.I. Giassetti, M. Ciccarelli, B. Lopez-Biladeau, J.M. Oatley, "Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation dagger" Biol. Reprod., vol. 101, pp. 177-187, 2019. [Crossref] [PubMed]

[111] M. Mehravar, A. Shirazi, M. Nazari, M. Banan, "Mosaicism in CRISPR/Cas9-mediated genome editing" Dev. Biol., vol. 445, pp. 156-162, 2019. [Crossref] [PubMed]

[112] R. Priyadharsini, R. Selvam, P. Satheeshkumar, P.N. Richard Jagatheesan, "A short glance about gene editing technology and its use in livestock" Int. J. Vet. Sci. Anim. Husb., vol. SP-8, pp. 37-40, 2023. Available at: https://www.veterinarypaper.com/archives/2023/8/2S/A/S-8-2-7.

[113] M. Hashimoto, Y. Yamashita, T. Takemoto, "Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse" Dev. Biol., vol. 418, pp. 1-9, 2016. [Crossref] [PubMed]

[114] E.V. O’Neil, K. Brooks, G.W. Burns, M.S. Ortega, A.C. Denicol, L.H. Aguiar, et al., "Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep" Mol. Reprod. Dev., vol. 87, pp. 142-151, 2020. [Crossref]

[115] I. Lamas-Toranzo, B. Galiano-Cogolludo, F. Cornudella-Ardiaca, J. Cobos-Figueroa, O. Ousinde, P. Bermejo-Alvarez, "Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos" Sci. Rep., vol. 9, 2019. [Crossref]

[116] X. Su, W. Chen, Q. Cai, P. Liang, Y. Chen, P. Cong, et al., "Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes" J. Genet. Genom., vol. 46, pp. 335-342, 2019. [Crossref]

[117] M. Chehelgerdi, M. Chehelgerdi, M. Khorramian-Ghahfarokhi, M. Shafieizadeh, E. Mahmoudi, F. Eskandari, et al., "Comprehensive review of CRISPR-based gene editing: Mechanisms, challenges, and applications in cancer therapy" Mol. Cancer, vol. 23, p. 9, 2024. [Crossref]

[118] E.M. Porto, A.C. Komor, "In the business of base editors: Evolution from bench to bedside" PLoS Biol., vol. 21, 2023. [Crossref]

[119] D.C. Choe, K. Musunuru, "Base editing: A brief review and a practical example" J. Biomed. Res., vol. 35, pp. 107-114, 2020. [Crossref]

[120] J. Kim, J.Y. Cho, J.-W. Kim, H.-C. Kim, J.K. Noh, Y.-O. Kim, et al., "CRISPR/Cas9-mediated myostatin disruption enhances muscle mass in the olive flounder Paralichthys olivaceus" Aquaculture, vol. 512, p. 734336, 2019. [Crossref]

[121] H.A. Rees, C. Wilson, J.L. Doman, D.R. Liu, "Analysis and minimization of cellular RNA editing by DNA adenine base editors" Sci. Adv., vol. 5, p. eaax5717, 2019. [Crossref] [PubMed]

[122] J. Xie, W. Ge, N. Li, Q. Liu, F. Chen, X. Yang, et al., "Efficient base editing for multiple genes and loci in pigs using base editors" Nat. Commun., vol. 10, p. 2852, 2019. [Crossref] [PubMed]

[123] M. Yuan, J. Zhang, Y. Gao, Z. Yuan, Z. Zhu, Y. Wei, et al., "HMEJ-based safe-harbor genome editing enables efficient generation of cattle with increased resistance to tuberculosis" J. Biol. Chem., vol. 296, 2021. [Crossref]

[124] R. Song, Y. Wang, Q. Zheng, J. Yao, C. Cao, Y. Wang, et al., "One-step base editing in multiple genes by direct embryo injection for pig trait improvement" Sci. China Life Sci., vol. 65, pp. 739-752, 2022. [Crossref]

[125] M.P. Zafra, E.M. Schatoff, A. Katti, M. Foronda, M. Breinig, A.Y. Schweitzer, et al., "Optimized base editors enable efficient editing in cells, organoids and mice" Nat. Biotechnol., vol. 36, pp. 888-893, 2018. [Crossref] [PubMed]

[126] Z. Liu, S. Chen, H. Shan, Y. Jia, M. Chen, Y. Song, et al., "Efficient base editing with high precision in rabbits using YFE-BE4max" Cell Death Dis., vol. 11, p. 36, 2020. [Crossref]

[127] Z. Huang, G. Liu, "Current advancement in the application of prime editing" Front. Bioeng. Biotechnol., vol. 11, 2023. [Crossref]

[128] J. Lee, K. Lim, A. Kim, Y.G. Mok, E. Chung, S.I. Cho, et al., "Prime editing with genuine Cas9 nickases minimizes unwanted indels" Nat. Commun., vol. 14, p. 1786, 2023. [Crossref]

[129] J. Zhang, M.L. Cui, Y.W. Nie, B. Dai, F.R. Li, D.J. Liu, et al., "CRISPR/Cas9-mediated specific integration of fat-1 at the goat MSTN locus" FEBS J., vol. 285, pp. 2828-2839, 2018. [Crossref]

[130] H.S. Kim, J. Kweon, Y. Kim, "Recent advances in CRISPR-based functional genomics for the study of disease-associated genetic variants" Exp. Mol. Med., vol. 56, pp. 861-869, 2024. [Crossref]

[131] R. Zhang, Y. Li, K. Jia, X. Xu, Y. Li, Y. Zhao, et al., "Crosstalk between androgen and Wnt/beta-catenin leads to changes of wool density in FGF5-knockout sheep" Cell Death Dis., vol. 11, p. 407, 2020. [Crossref] [PubMed]

[132] C. Dilger, X. Chen, L.T. Honegger, B.M. Marron, J.E. Beever, "The potential for gene-editing to increase muscle growth in pigs: Experiences with editing myostatin" CABI Agric. Biosci., vol. 3, 2022. [Crossref]

[133] H. Güralp, K. Skaftnesmo, E. Kjærner-Semb, A.H. Straume, L. Kleppe, R.W. Schulz, et al., "Rescue of germ cells in Dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon" Sci. Rep., vol. 10, 2020. [Crossref]

[134] O. Matika, D. Robledo, R. Pong-Wong, S.C. Bishop, V. Riggio, H. Finlayson, et al., "Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs" PLOS Genet., vol. 15, 2019. [Crossref]

[135] C. Proudfoot, D.F. Carlson, R. Huddart, C.R. Long, J.H. Pryor, T.J. King, et al., "Genome edited sheep and cattle" Transgenic Res., vol. 24, pp. 147-153, 2015. [Crossref]

[136] R. Guo, Y. Wan, D. Xu, L. Cui, M. Deng, G. Zhang, et al., "Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system" Sci. Rep., vol. 6, 2016. [Crossref] [PubMed]

[137] M.L. Mueller, A.L. Van Eenennaam, "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle" CABI Agric. Biosci., vol. 3, 2022. [Crossref]

[138] E.J. Loe, "Towards a more robust salmon: Using CRISPR/Cas9 editing to increase protein levels in Zebrafish and Atlantic salmon" Master Thesis, Department of Biological Sciences, University of Bergen, Bergen, Norway, 2024.

[139] K.A. Glover, M.F. Solberg, P. McGinnity, K. Hindar, E. Verspoor, M.W. Coulson, et al., "Half a century of genetic interaction between farmed and wild Atlantic salmon: Status of knowledge and unanswered questions" Fish, vol. 18, pp. 890-927, 2017. [Crossref]

[140] A. Wargelius, S. Leininger, K.O. Skaftnesmo, L. Kleppe, E. Andersson, G.L. Taranger, et al., "Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon" Sci. Rep., vol. 6, 2016. [Crossref]

[141] L. Kleppe, E. Andersson, K.O. Skaftnesmo, R.B. Edvardsen, P.G. Fjelldal, B. Norberg, et al., "Sex steroid production associated with puberty is absent in germ cell-free salmon" Sci. Rep., vol. 7, 2017. [Crossref]

[142] Z. Qin, Y. Li, B. Su, Q. Cheng, Z. Ye, D.A. Perera, et al., "Editing of the luteinizing hormone gene to sterilize Channel Catfish, Ictalurus punctatus, using a modified zinc finger nuclease technology with electroporation" Mar. Biotechnol., vol. 18, pp. 255-263, 2016. [Crossref]

[143] Y. Wu, T. Wu, L. Yang, Y. Su, C. Zhao, L. Li, et al., "Generation of fast growth Nile tilapia (Oreochromis niloticus) by myostatin gene mutation" Aquaculture, vol. 562, p. 738762, 2023. [Crossref]

[144] Z. Zhong, P. Niu, M. Wang, G. Huang, S. Xu, Y. Sun, et al., "Targeted disruption of Sp7 and Myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common Carp" Sci. Rep., vol. 6, 2016. [Crossref] [PubMed]

[145] K. Khalil, M. Elayat, E. Khalifa, S. Daghash, A. Elaswad, M. Miller, et al., "Generation of Myostatin gene-edited channel catfish (Ictalurus punctatus) via zygote injection of CRISPR/Cas9 system" Sci. Rep., vol. 7, 2017. [Crossref]

[146] K.I. Kishimoto, Y. Washio, Y. Yoshiura, A. Toyoda, T. Ueno, H. Fukuyama, et al., "Production of a breed of red sea bream Pagrus major with an increase of skeletal muscle mass and reduced body length by genome editing with CRISPR/Cas9" Aquaculture, vol. 495, pp. 415-427, 2018. [Crossref]

[147] H. Yu, H. Li, Q. Li, R. Xu, C. Yue, S. Du, "Targeted gene disruption in Pacific Oyster based on CRISPR/Cas9 ribonucleoprotein complexes" Mar. Biotechnol., vol. 21, pp. 301-309, 2019. [Crossref] [PubMed]

[148] X.L. Su, G.D. Zheng, S.M. Zou, "Knockout of EPO gene in blunt snout bream (Megalobrama amblycephala) by CRISPR/Cas9 reveals its roles in hypoxia-tolerance" Aquaculture, vol. 592, p. 741227, 2024. [Crossref]

[149] M. Ohama, Y. Washio, K. Kishimoto, M. Kinoshita, K. Kato, "Growth performance of Myostatin knockout Red Sea bream Pagrus Major juveniles produced by genome editing with CRISPR/Cas9" Aquaculture, vol. 529, p. 735672, 2020. [Crossref]

[150] B. Tao, J. Tan, L. Chen, Y. Xu, X. Liao, Y. Li, et al., "CRISPR/Cas9 System-Based Myostatin-Targeted Disruption Promotes Somatic Growth and Adipogenesis in Loach, Misgurnus Anguillicaudatus" Aquaculture, vol. 544, p. 737097, 2021. [Crossref]

[151] Y. ShimbunY. Shimbun, "Kyoto Firm Puts Genome-Edited Tiger Pufer on the Table" Japan News Available online: https://the-japan-news.com/news/article/000793605. (accessed on 1 November 2021)

[152] S.K. Panda, M.J. McGrew, "Genome editing of avian species: Implications for animal use and welfare" Lab. Anim., vol. 56, pp. 50-59, 2022. [Crossref] [PubMed]

[153] J.S. Park, K.Y. Lee, J.Y. Han, "Precise genome editing in poultry and its application to industries" Genes, vol. 11, 2020. [Crossref]

[154] M.C. Van de Lavoir, J.H. Diamond, P.A. Leighton, C. Mather-Love, B.S. Heyer, R. Bradshaw, et al., "Germline transmission of genetically modified primordial germ cells" Nature, vol. 441, pp. 766-769, 2006. [Crossref] [PubMed]

[155] G. Kim, J.H. Lee, S. Song, S.W. Kim, J.S. Han, S.P. Shin, et al., "Generation of myostatin-knock-out chickens mediated by D10A-Cas9 nickase" FASEB J., vol. 34, pp. 5688-5696, 2020. [Crossref]

[156] B. Schusser, E.J. Collarini, H. Yi, S.M. Izquierdo, J. Fesler, D. Pedersen, et al., "Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells" Proc. Natl. Acad. Sci. USA, vol. 110, pp. 20170-20175, 2013. [Crossref]

[157] T.S. Park, H.J. Lee, K.H. Kim, J. Kim, J.Y. Han, "Targeted gene knock-out in chickens mediated by TALENs" Proc. Natl. Acad. Sci., vol. 111, pp. 12716-21, 2014. [Crossref] [PubMed]

[158] I. Oishi, K. Yoshii, D. Miyahara, T. Tagami, "Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens" Sci. Rep., vol. 8, 2018. [Crossref]

[159] T.S. Park, J. Park, J.H. Lee, J. Park, B. Park, "Disruption of G0/G 1 switch gene 2 (G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken" FASEB J., vol. 33, pp. 1188-98, 2019. [Crossref]

[160] T.S. Park, "Gene-editing techniques and their applications in livestock and beyond" Anim Biosci, vol. 36, pp. 333-338, 2023. [Crossref]

[161] J. Lee, D.H. Kim, K. Lee, "Myostatin gene role in regulating traits of poultry species for potential industrial applications" J. Anim. Sci. Biotechnol., vol. 15, 2024. [Crossref]

[162] K. Xu, C.X. Han, H. Zhou, J.M. Ding, Z. Xu, L.Y. Yang, et al., "Effective MSTN gene knockout by AdV-delivered CRISPR/Cas9 in postnatal chick leg muscle" Int. J. Mol. Sci., vol. 21, 2020. [Crossref] [PubMed]

[163] M. Watanabe, K. Umeyama, H. Matsunari, S. Takayanagi, E. Haruyama, K. Nakano, et al., "Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases" Biochem. Biophys. Res. Commun., vol. 402, pp. 14-18, 2010. [Crossref]

[164] J. Whyte, J. Zhao, K. Wells, M. Samuel, K. Whitworth, E. Walters, et al., "Gene targeting with Zinc Finger Nucleases to produce cloned eGFP knockout pigs" Mol Reprod Dev, vol. 78, p. 10, 2011. [Crossref]

[165] L. Qian, M. Tang, J. Yang, Q. Wang, C. Cai, S. Jiang, et al., "Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs" Sci. Rep., vol. 5, 2015. [Crossref]

[166] S. Rao, T. Fujimura, H. Matsunari, T. Sakuma, K. Nakano, M. Watanabe, et al., "Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets" Mol. Reprod. Dev., vol. 83, pp. 61-70, 2016. [Crossref] [PubMed]

[167] K. Wang, X. Tang, Z. Xie, X. Zou, M. Li, H. Yuan, et al., "CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs" Transgenic Res., vol. 26, pp. 799-805, 2017. [Crossref] [PubMed]

[168] Y. Zou, Z. Li, Y. Zou, H.-Y. Hao, J.-X. Hu, N. Li, et al., "Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination" J. Integrative Agr., vol. 18, pp. 1329-1336, 2019. [Crossref]

[169] M. Hirata, M. Wittayarat, Z. Namula, Q.A. Le, Q. Lin, K. Takebayashi, et al., "Generation of mutant pigs by lipofection-mediated genome editing in embryos" Sci. Rep., vol. 11, 2021. [Crossref]

[170] Z. Fan, Z. Liu, K. Xu, T. Wu, J. Ruan, X. Zheng, et al., "Long-term, multidomain analyses to identify the breed and allelic effects in MSTN-edited pigs to overcome lameness and sustainably improve nutritional meat production" Sci. China Life Sci., vol. 65, pp. 362-375, 2022. [Crossref]

[171] D. Preisinger, T. Winogrodzki, B. Klinger, A. Schnieke, B. Rieblinger, "Genome editing in pigs" Methods Mol. Biol., vol. 2631, pp. 393-417, 2023. [Crossref] [PubMed]

[172] Y. Zou, Z. Li, Y. Zou, H. Hao, N. Li, Q. Li, "An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects" Biochem. Biophys. Res. Commun., vol. 498, pp. 940-945, 2018. [Crossref] [PubMed]

[173] G. Xiang, J. Ren, T. Hai, R. Fu, D. Yu, J. Wang, et al., "Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs" Cell Mol. Life Sci., vol. 75, pp. 4619-4628, 2018. [Crossref]

[174] A. Jabbar, F. Zulfiqar, M. Mahnoor, N. Mushtaq, M.H. Zaman, A.S.U. Din, et al., "Advances and perspectives in the application of CRISPR-Cas9 in livestock" Mol. Biotechnol., vol. 63, pp. 757-767, 2021. [Crossref] [PubMed]

[175] S. Younis, M. Schönke, J. Massart, R. Hjortebjerg, E. Sundström, U. Gustafson, et al., "The ZBED6–IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals" Proc. Natl. Acad. Sci. USA, vol. 115, pp. E2048-E2057, 2018. [Crossref] [PubMed]

[176] X. Liu, H. Liu, M. Wang, R. Li, J. Zeng, D. Mo, et al., "Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs" Transgenic Res., vol. 28, pp. 141-150, 2019. [Crossref]

[177] B.M. Ehn, T. Allmere, E. Telemo, U. Bengtsson, B. Ekstrand, "Modification of IgE binding to beta-lactoglobulin by fermentation and proteolysis of cow’s milk" J. Agric. Food Chem., vol. 53, pp. 3743-3748, 2005. [Crossref]

[178] Z. Sun, M. Wang, S. Han, S. Ma, Z. Zou, F. Ding, et al., "Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA" Sci. Rep., vol. 8, 2018. [Crossref]

[179] J. Wei, S. Wagner, P. Maclean, B. Brophy, S. Cole, G. Smolenski, et al., "Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin" Sci. Rep., vol. 8, 2018. [Crossref]

[180] Y.Y. Silaeva, M.V. Kubekina, A.V. Bruter, A.G. Isaeva, A.G. Koshchaev, "Gene editing CRISPR/Cas9 system for producing cows with hypoallergenic milk on the background of a beta-lacto globulin gene knockout" E3S Web Conf. (IDSISA), vol. 176, p. 01006, 2020. [Crossref]

[181] J. Luo, Z. Song, S. Yu, D. Cui, B. Wang, F. Ding, et al., "Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases" PLoS ONE, vol. 9, 2014. [Crossref]

[182] K. Simmet, V. Zakhartchenko, J. Philippou-Massier, H. Blum, N. Klymiuk, E. Wolf, "OCT4/POU5F1 is required for NANOG expression in bovine blastocysts" Proc. Natl. Acad. Sci. USA, vol. 115, pp. 2770-2775, 2018. [Crossref] [PubMed]

[183] L.S.A. Camargo, J.F. Pereira, "Genome-editing opportunities to enhance cattle productivity in the tropics" CABI Agric. Biosci., vol. 3, 2022. [Crossref]

[184] D.F. Carlson, C.A. Lancto, B. Zang, E.-S. Kim, M. Walton, D. Oldeschulte, et al., "Production of hornless dairy cattle from genome-edited cell lines" Nat. Biotechnol., vol. 34, pp. 479-481, 2016. [Crossref] [PubMed]

[185] M.J. Gu, L. Gao, X.Y. Zhou, D. Wu, Z.Y. Wei, G.P. Li, et al., "Fixed-point editing of POLLED sites in Mongolian cattle without horns" J. Agric. Biotechnol., vol. 28, pp. 242-250, 2020.

[186] F. Schuster, P. Aldag, A. Frenzel, K.-G. Hadeler, A. Lucas-Hahn, H. Niemann, et al., "CRISPR/Cas12a mediated knock-in of the polled celtic variant to produce a polled genotype in dairy cattle" Sci. Rep., vol. 10, 2020. [Crossref]

[187] G. Laible, S.A. Cole, B. Brophy, J. Wei, S. Leath, S. Jivanji, et al., "Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change" BMC Genomics, vol. 22, 2021. [Crossref]

[188] F. Sosa, J.E.P. Santos, D.O. Rae, C.C. Larson, M. Macchietto, J.E. Abrahante, et al., "Effects of the SLICK1 mutation in PRLR on regulation of core body temperature and global gene expression in liver in cattle" Animal, vol. 16, p. 100523, 2022. [Crossref]

[189] A.T. Carmickle, C.C. Larson, F.S. Hernandez, J.M.V. Pereira, F.C. Ferreira, M.L.J. Haimon, et al., "Physiological responses of Holstein calves and heifers carrying the SLICK1 allele to heat stress in California and Florida dairy farms, J" Dairy Sci., vol. 105, pp. 9216-9225, 2022. [Crossref]

[190] L.R. Porto-Neto, D.M. Bickhart, A.J. Landaeta-Hernandez, Y.T. Utsunomiya, M. Pagan, E. Jimenez, et al., "Convergent evolution of Slick Coat in cattle through truncation mutations in the prolactin receptor" Front. Genet., vol. 9, 2018. [Crossref]

[191] M.-Y. Qi, L.-Q. Xu, J.-N. Zhang, M.-O. Li, M.-H. Lu, Y.-C. Yao, "Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction" Theriogenology, vol. 142, pp. 246-250, 2020. [Crossref]

[192] P. Kalds, S. Zhou, B. Cai, J. Liu, Y. Wang, B. Petersen, et al., "Sheep and goat genome engineering: From random transgenesis to the CRISPR era" Front. Genet., vol. 10, 2019. [Crossref] [PubMed]

[193] Y. Niu, X. Zhao, J. Zhou, Y. Li, Y. Huang, B. Cai, et al., "Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9" Reprod. Fertil. Dev., vol. 30, p. 307, 2018. [Crossref] [PubMed]

[194] X. Tian, D. Lv, T. Ma, S. Deng, M. Yang, Y. Song, et al., "AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring" Peer J., vol. 6, p. e5420, 2018. [Crossref] [PubMed]

[195] H. Li, G. Wang, Z. Hao, G. Zhang, Y. Qing, S. Liu, et al., "Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer" Sci. Rep., vol. 6, 2016. [Crossref]

[196] Z. He, T. Zhang, L. Jiang, M. Zhou, D. Wu, J. Mei, et al., "Use of CRISPR/Cas9 technology efficiently targeted goat myostatin through zygotes microinjection resulting in double-muscled phenotype in goats" Biosci. Rep., vol. 38, 2018. [Crossref]

[197] H. Han, Y. Ma, T. Wang, L. Lian, X. Tian, R. Hu, et al., "One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system" Front. Agr. Sci. Eng., vol. 1, pp. 2-5, 2014. [Crossref]

[198] H.T. Wang, T.T. Li, X. Huang, R.L. Ma, Q.Y. Liu, "Application of genetic modification technologies in molecular design breeding of sheep" Yi Chuan Hered., vol. 43, pp. 580-600, 2021. [Crossref] [PubMed]

[199] Y. Zhang, Y. Wang, B. Yulin, B. Tang, M. Wang, C. Zhang, et al., "CRISPR/Cas9-mediated sheep MSTN gene knockout and promote sSMSCs differentiation" J. Cell Biochem., vol. 120, pp. 1794-1806, 2019. [Crossref]

[200] S. Zhou, P. Kalds, Q. Luo, K. Sun, X. Zhao, Y. Gao, et al., "Optimized Cas9: sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality" BMC Genom., vol. 23, 2022. [Crossref]

[201] J.Y. Mei, "Targeted Knockout of MSTN Gene in Goats Using Crispr/Cas9 Technology" Master’s Thesis, Yangzhou University, Yangzhou, China, 2017.

[202] C. Cui, Y. Song, J. Liu, H. Ge, Q. Li, H. Huang, et al., "Gene targeting by TALEN-induced homologous recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk" Sci. Rep., vol. 5, 2015. [Crossref]

[203] T. Ma, J. Tao, M. Yang, C. He, X. Tian, X. Zhang, et al., "An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep" J. Pineal Res., vol. 63, p. e12406, 2017. [Crossref]

[204] W. Zhou, Y. Wan, R. Guo, M. Deng, K. Deng, Z. Wang, et al., "Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9" PLoS ONE, vol. 12, 2017. [Crossref] [PubMed]

[205] H. Tian, J. Luo, Z. Zhang, J. Wu, T. Zhang, S. Busato, et al., "CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) deficiency affects fatty acid metabolism in goat mammary epithelial cells" J. Agric. Food Chem., vol. 66, pp. 10041-10052, 2018. [Crossref] [PubMed]

[206] Y. Zhang, Y. Wang, X. Wan, Y. Ji, S. Cheng, M. Wang, et al., "Acetyl-coenzyme A acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes" J. Cell Biochem., vol. 120, pp. 8021-8031, 2019. [Crossref] [PubMed]

[207] K. Xu, Y. Zhou, Y. Mu, Z. Liu, S. Hou, Y. Xiong, et al., "CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance" eLife, vol. 9, p. e57132, 2020. [Crossref]

[208] X. Wang, B. Cai, J. Zhou, H. Zhu, Y. Niu, B. Ma, et al., "Disruption of FGF5 in Cashmere goats using CRISPR/Cas9 results in more secondary hair follicles and longer fibers" PLoS ONE, vol. 11, 2016. [Crossref]

[209] F. Hao, W. Yan, X. Li, H. Wang, Y. Wang, X. Hu, et al., "Generation of Cashmere goats carrying an EDAR gene mutant using CRISPR-Cas9-mediated genome editing" Int. J. Biol. Sci., vol. 14, pp. 427-436, 2018. [Crossref]

[210] B. Dai, R.N. Sha, J.L. Yuan, D.J. Liu, "Multiple potential roles of thymosin β4 in the growth and development of hair follicles" J. Cell Mol. Med., vol. 25, pp. 1350-1358, 2021. [Crossref]

[211] X. Wang, Y. Niu, J. Zhou, H. Yu, Q. Kou, A. Anmin Lei, et al., "Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep" Sci. Rep., vol. 6, 2016. [Crossref]

[212] C. Proudfoot, S. Lillico, C. Tait-Burkard, "Genome editing for disease resistance in pigs and chickens" Anim. Front., vol. 9, pp. 6-12, 2019. [Crossref]

[213] M. Tian, X. He, Y. Feng, W. Wang, H. Chen, M. Gong, et al., "Pollution by antibiotics and antimicrobial resistance in livestock and poultry manure in China, and countermeasures" Antibiotics, vol. 10, 2021. [Crossref]

[214] J.P. Wang, H.Z. Lu, T. Zhang, L. Wang, "Research progress on the application of CRISPR/Cas9 technology in chicken antiviral infection" Heilongjiang Anim. Sci. Vet. Med., pp. 28-34, 2022.

[215] C.-F. Tu, C.-K. Chuang, K.-H. Hsiao, C.-H. Chen, C.-M. Chen, S.-H. Peng, et al., "Lessening of porcine epidemic diarrhea 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, vol. 14, 2019. [Crossref]

[216] A.S. Madhun, R. Nilsen, B.T. Barlaup, Ø. Karlsen, E. Karlsbakk, "Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway" Fish. Dis., vol. 47, p. e13874, 2024. [Crossref] [PubMed]

[217] V. Chakrapani, S. Patra, R.P. Panda, K.D. Rasal, P. Jayasankar, H.K. Barman, "Establishing targeted Carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9" Dev. Comp. Immunol., vol. 61, pp. 242-247, 2016. [Crossref] [PubMed]

[218] J. Ma, Y. Fan, Y. Zhou, W. Liu, N. Jiang, J. Zhang, et al., "Efficient resistance to Grass Carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9" Fish Shellfish Immunol., vol. 76, pp. 206-215, 2018. [Crossref]

[219] M. Matoušková, J. Plachý, D. Kučerová, Ľ. Pecnová, M. Reinišová, J. Geryk, et al., "Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance" PLoS Pathog., vol. 20, 2024. [Crossref] [PubMed]

[220] Y.H. Park, S.J. Woo, K. Chungu, S.B. Lee, J.H. Shim, H.J. Lee, et al., "Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase" FASEB J., vol. 35, p. e21630, 2021. [Crossref]

[221] T. Lu, B. Yang, R. Wang, C. Qin, "Xenotransplantation: Current status in preclinical research" Front. Immunol., vol. 10, 2019. [Crossref]

[222] K.D. Wells, R. Bardot, K.M. Whitworth, B.R. Trible, Y. Fang, A. Mileham, et al., "Replacement of porcine CD163 scavenger receptor cysteine-rich domain 5 with a CD163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus" J. Virol., vol. 91, p. e01521-16, 2017. [Crossref]

[223] N.J. Boddicker, A. Bjorkquist, R.R. Rowland, J.K. Lunney, J.M. Reecy, J.C. Dekkers, "Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection" Genet. Sel. Evol., vol. 46, p. 18, 2014. [Crossref] [PubMed]

[224] J.G. Calvert, D.E. Slade, S.L. Shields, R. Jolie, R.M. Mannan, R.G. Ankenbauer, et al., "CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses" J. Virol., vol. 81, pp. 7371-7379, 2007. [Crossref]

[225] K.M. Whiteworth, R.R. Rowland, V. Petrovan, M. Sheahan, A.G. Cino-Ozuna, Y. Fang, et al., "Resistance to coronavirus infection in amino peptidase N-deficient pigs" Transgenic Res., vol. 28, pp. 21-32, 2019. [Crossref]

[226] C. Burkard, T. Opriessnig, A.J. Mileham, T. Stadejek, T. Ait-Ali, S.G. Lillico, et al., "Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection" J. Virol., vol. 92, pp. JVI.00415-00418, 2018. [Crossref]

[227] H. Wang, L. Shen, J. Chen, X. Liu, T. Tan, Y. Hu, et al., "Deletion of CD163 Exon 7 confers resistance to highly pathogenic porcine reproductive and respiratory viruses on pigs" Int. J. Biol. Sci., vol. 15, pp. 1993-2005, 2019. [Crossref] [PubMed]

[228] L. Luo, S. Wang, L. Zhu, B. Fan, T. Liu, L. Lefeng Wang, et al., "Aminopeptidase N-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus" Sci. Rep., vol. 9, 2019. [Crossref] [PubMed]

[229] H. Yang, J. Zhang, X. Zhang, J. Shi, Y. Pan, R. Zhou, et al., "CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus" Antivir. Res., vol. 151, pp. 63-70, 2018. [Crossref]

[230] A.M. Cigan, P.W. Knap, "Technical considerations towards commercialization of porcine respiratory and reproductive syndrome (PRRS) virus resistant pigs" CABI Agric. Biosci., vol. 3, 2022. [Crossref]

[231] A. Stoian, R.R. Rowland, V. Petrovan, M. Sheahan, M.S. Samuel, K.M. Whitworth, et al., "The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine delta coronavirus (PDCoV)" Virology, vol. 541, pp. 136-140, 2020. [Crossref]

[232] H. Wu, Y. Wang, Y. Zhang, M. Yang, J. Lv, J. Liu, et al., "TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis" Proc. Natl. Acad. Sci. USA, vol. 112, pp. E1530-E1539, 2015. [Crossref]

[233] , "Program and Abstracts of the 14th Transgenic Technology Meeting (TT2017): Snowbird Resort, Salt Lake City, Utah, USA, 1–4 October 2017" Transgenic Res., vol. 26, pp. 1-45, 2017. [Crossref] [PubMed]

[234] A. Hübner, B. Petersen, G.M. Keil, H. Niemann, T.C. Mettenleiter, W. Fuchs, "Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L)" Sci. Rep., vol. 8, 2018. [Crossref]

[235] X.J. Li, Y.H. He, X.Y. Zhu, X. Zou, C.L. Luo, "Research progress on the application of CRISPR/Cas9 technology in pigs and chickens" Chin. Anim. Husb. Vet. Med., vol. 49, pp. 4665-4673, 2022. Available at: https://www.chvm.net/EN/10.16431/j.cnki.1671-7236.2022.12.016.

[236] J.W.M. Bastiaansen, H. Bovenhuis, M.A.M. Groenen, H.-J. Megens, H.A. Mulder, "The impact of genome editing on the introduction of monogenic traits in livestock" Genet. Sel. Evol., vol. 5, p. 18, 2018. [Crossref]

[237] G. Reiner, "Genetic resistance - an alternative for controlling PRRS?" Porc. Health Manag., vol. 2, p. 27, 2016. [Crossref]

[238] J. Dukes, J. Wrathall, P. Hawkins, E. Coughlan, S. CollinsJ. Dukes, J. Wrathall, P. Hawkins, E. Coughlan, S. Collins, "RSPCA Response to Genome Editing and Farmed Animals, Nuffield Council for Bioethics Call for Evidence, September 2019. Science and Policy Group, RSPCA. 77d17c92-0bc5-6eb0-1837-1bc430fbd97f" Available online: https://www.rspca.org.uk/documents/1494939/7712578/RSPCA+response+-+Genome+Editing+and+farmed+animals.pdf/77d17c92-0bc5-6eb0-1837-1bc430fbd97f?t=1584525808480.

[239] S. Shanthalingam, A. Tibary, J.E. Beever, P. Kasinathan, W.C. Brown, S. Srikumaran, "Precise gene editing paves the way for derivation of Mannheimia haemolytica leukotoxin-resistant cattle" Proc. Natl. Acad. Sci. USA, vol. 113, pp. 13186-13190, 2016. [Crossref] [PubMed]

[240] Y. Gao, H. Wu, Y. Wang, X. Liu, L. Chen, Q. Li, et al., "Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects" Genome Biol., vol. 18, 2017. [Crossref]

[241] A. Pal, A.K. Chakravarty, "Disease resistance for different livestock species" Genet. Breed. Dis. Resist. Livestock, pp. 271-296, 2020. [Crossref]

[242] K.P. Szillat, S. Koethe, K. Wernike, D. Höper, M. Beer, "A CRISPR/Cas9 generated bovine CD46-knockout cell line-A tool to elucidate the adaptability of bovine viral diarrhea viruses (BVDV)" Viruses, vol. 12, 2020. [Crossref]

[243] A.M. Workman, M.P. Heaton, B.L. Vander Ley, D.A. Webster, L. Sherry, J.R. Bostrom, et al., "First gene-edited calf with reduced susceptibility to a major viral pathogen" PNAS Nexus, vol. 2, pp. 1-14, 2023. [Crossref]

[244] X. Liu, Y. Wang, Y. Tian, Y. Yu, M. Gao, G. Hu, et al., "Generation of mastitis resistance in cows by targeting human lysozyme gene to beta-casein locus using zinc-finger nucleases" Proc. Biol. Sci., vol. 281, 2014. [Crossref]

[245] J.F. O’Toole, W. Schilling, D. Kunze, S.M. Madhavan, M. Konieczkowski, Y. Gu, et al., "ApoL1 overexpression drives variant-independent cytotoxicity" J. Am. Soc. Nephrol., vol. 29, pp. 869-879, 2018. [Crossref]

[246] M. KarembuM. Karembu, "Genome Editing in Africa’s Agriculture 2021: An Early Take-Of. Nairobi, Kenya: International Service for the Acquisition of Agri-Biotech Applications (ISAAA AfriCenter)" Available online: https://africenter.isaaa.org/wp-content/uploads/2021/04/GENOME-EDITING-IN-AFRICA-FINAL.pdf.

[247] D.K. Williams, C. Pinzón, S. Huggins, J.H. Pryor, A. Falck, F. Herman, et al., "Genetic engineering a large animal model of human hypophosphatasia in sheep" Sci. Rep., vol. 8, 2018. [Crossref] [PubMed]

[248] A. Menchaca, P.C. Dos Santos-Neto, M. Souza-Neves, F. Cuadro, A.P. Mulet, L. Tesson, et al., "Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated homology directed repair" Sci. Rep., vol. 10, 2020. [Crossref] [PubMed]

[249] Z. Fan, M. Yang, M. Regouski, I.A. Polejaeva, "Gene knockouts in goats using CRISPR/Cas9 system and somatic cell nuclear transfer" Methods Mol. Biol., vol. 1874, pp. 373-390, 2019. [Crossref] [PubMed]

[250] M. Vilarino, F.P. Suchy, S.T. Rashid, H. Lindsay, J. Reyes, B.R. McNabb, et al., "Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep" Transgenic Res., vol. 27, pp. 525-537, 2018. [Crossref]

[251] M.J. Landrum, J.M. Lee, M. Benson, G. Brown, C. Chao, S. Chitipiralla, et al., "ClinVar: Public archive of interpretations of clinically relevant variants" Nucleic Acids Res., vol. 44, pp. D862-D868, 2016. [Crossref]

[252] T. Feng, Z. Li, X. Qi, J. Liu, F. Gao, Z. Ma, et al., "Measuring targeting specificity of genome-editing by nuclear transfer and sequencing (NT-seq)" Cell Discov., vol. 6, p. 78, 2020. [Crossref]

[253] E. Collins, "FDA approves antithrombin ATryn from genetically altered animals" Wash. Drug Lett., vol. 41, p. 10, 2009.

[254] C. Sheridan, "FDA approves “farmaceutical” drug from transgenic chickens" Nat. Biotechnol., vol. 34, pp. 117-119, 2016. [Crossref]

[255] "FDA Approves Genetically Modified salmon for Human Consumption" Available online: https://www.labmanager.com/fda-approves-genetically-modified-salmon-for-human-consumption-10978. (accessed on 19 November 2015)