Striking examples of pioneering experiments in obtaining transgenic farm animals are the knock-out pig and ferret models for CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). These larger CFTR animal models recapitulate many phenotypic characteristics of the human disease, which are clinically much more relevant than those found in the corresponding knock-out mice
[18]. Increasing weight gain in beef cattle and pigs is the top priority for the industry. One example of the superior efficiency of transgenic animals in production is GM gilts that produce 70% more milk than the control non-transgenic littermates. The offspring of these gilts will grow 500 g larger in 21 days of lactation
[19]. As another example, Dr. Alison Van Eenennaam’s team proposes a genome-engineered solution to produce hornless cow breeds with superior muscle mass
[20][21]. An alternative approach to increase productivity is to manipulate the reproductive performance of livestock. Thus, one study involved the introduction of a point mutation in the
GDF9 (growth differentiation factor 9) gene, which had a large influence on both the rate of ovulation and the size of the litter in goats
[22]. Likewise, another study showed that a mutation in the
BMPR-1B (
FecB) gene in sheep leads to an increased rate of ovulation and, consequently, to an increase in litter size
[23].
2. Precision Genome-Editing Tools Successfully Used in Livestock
2.1. Zinc Finger Nucleases (ZFNs)
Zinc finger nucleases (ZFNs) are engineered nucleases containing a zinc finger protein domain as a DNA binding component and a DNA cleavage domain fused together to form an artificial restriction enzyme
[34]. Zinc finger domains are designed to bind specific nucleotide combinations within DNA. This allows ZFNs to target unique sequences within complex genomes. Each zinc finger domain binds three-nucleotide sequences in a sequence-specific manner. Combining several “zinc finger” domains allows ZFNs to recognize virtually any DNA sequence. The most commonly used nuclease is the catalytic domain of the restriction enzyme
FokI, which consists of two subunits, so nucleases work in pairs. The recognition sites are chosen so that the distances between them are sufficient to dimerize
FokI domains and form a catalytically active structure
[34].
The main allergen found in goat and cow milk is β-lactoglobulin, which is not present in human milk. Genetic engineers had a long-term objective of producing milk from animal sources that is depleted of β-lactoglobulin and contains a biologically active human protein. The task was to “humanize” goat milk by integrating human milk protein genes, lactoferrin or lactalbumin, into the endogenous gene region. ZFN technology was used to solve this long-standing issue by creating gene-edited cattle in the β-lactoglobulin locus
[35].
Other successful examples of applied ZFN technology in farm animals involve pigs and cows. For example,
GGTA1 (α-1,3-galactosyltransferase) was successfully knocked out in pigs—the first step in creating transgenic swine donors
[36]. Likewise, Liu and co-authors used a ZFN to produce transgenic cows carrying the human lysozyme gene in the bovine β-casein locus
[37]. Such animals are resistant to mastitis since the milk secreted by transgenic cows has the ability to inactivate
Staphylococcus aureus [2].
Injection of ZFN mRNA or DNA into zygotes has been successfully used to edit the genome in rabbits and rodents
[38][39]. ZFN in-embryo editing helped to efficiently achieve interspecies allele introgression in one generation in pigs
[40]. This method has several disadvantages, including the possibility of off-target cleavage by zinc finger domains. The method is also labor-intensive, as it requires creating a ZFN protein structure for every DNA sequence. Thus, the “zinc finger” system has not been widely adopted
[10].
2.2. Transcription Activator-like Effector Nucleases (TALENs)
Another relevant and widely used method of genome editing of farm animals today is based on chimeric nucleases called transcription activator-like effector nucleases (TALENs)
[41][42]. The TAL protein domains are responsible for the recognition of specific nucleotides and can be combined to create a sequence-specific binding subunit, which in turn is fused to a DNA cleavage domain of a
FokI endonuclease. Such proteins usually contain a DNA-binding domain consisting of 33–35 consecutive amino acid repeats that specifically bind to the host’s genomic DNA
[43]. TAL-domain prototype proteins come from
Xanthomonas bacteria where they facilitate plant infection
[34]. In 2011, this approach was awarded a “Method of the Year” award in
Nature Methods due to the wide range of possible applications in the fields of basic and applied science, from functional genomics to developmental biology and agricultural biotechnology
[44].
The development of TALEN technology facilitated the knock-in strategy, the essence of which is the integration of target genes into specific regions of the genome. This strategy has been used to replace
BSA (bovine serum albumin) with two
hSA (human serum albumin) minigenes to specifically express them in the liver and mammary gland in cattle
[45]. The knock-in strategy appeared as a promising way to create cows producing recombinant therapeutic proteins in milk
[41].
In 2015, TALEN technology was used to perform the genetic editing of bulls
[20]. A
POLLED mutation has been introduced, which is sometimes found in the wild and is associated with the lack of horns. For agricultural breeds, this mutation could be a valuable asset, as horns make animals more dangerous to personnel. Now, bulls have their horns removed, but it was considered more cost-effective and convenient to create a hornless bull breed. Researchers chose TALEN technology to edit genomes in the connective tissue cells of an adult bull. Then, the nuclei were collected and transferred to the eggs of cows by reproductive cloning
[20]. Amy Young and colleagues tested the genome of the edited bulls and evaluated the efficiency of the new mutation’s transmission to the next generation
[21]. As a result, the edited bull produced six offspring, all hornless. All six calves were heterozygotes, carrying one wild-type and one dominant mutant allele. DNA testing showed that no traces of TALEN off-target activity, i.e., no unplanned mutations, were found. The offspring showed no physiological abnormalities, or any health disorders, with the exception of one bull whose testicle did not descend. The remaining four were tested according to all veterinary standards and were recognized as potential sires
[21]. It was also shown that GM offspring had no effect on the mothers. Microchimerism in mammals is common when fetal cells colonize the red bone marrow of the mother. Scientists could not detect any mutant genes in the cows’ blood, either during or after pregnancy. They confirmed that genetic modification did not pass from fetuses to mothers in any way
[46].
However, despite some advances in the production of GM animals, TALEN technology has a number of limitations. Its high cost, combined with the difficulty of constructing recombinant vectors, makes it a less attractive option than traditional knock-out technologies.
2.3. Clustered Regularly Interspaced Palindromic Repeats (CRISPR)
New opportunities have appeared following the introduction of the new genome-editing system, which revolutionized the production of GM animals. Le Cong described the first use of the CRISPR/Cas9 system and brought genetic engineering to a new level
[47].
The mechanism that bacteria use to defend themselves against their pathogenic viruses (bacteriophages) inspired the development of this system
[48]. The Cas9 protein makes a double-strand break around the protospacer adjacent motif (PAM), which contains a conserved NGG sequence. Cas9 navigates to the PAM using guide RNA complementary to a 19-nucleotide sequence upstream of the PAM
[49]. The non-homologous end-joining repair machinery introduces small deletions or inserts at the site of the break, thereby creating possible mutations. Simultaneous addition of constructs that contain homology arms around the break results in homologous recombination. This facilitates the insertion of the desired fragment at a specific location in the genome. The CRISPR/Cas9 technology has the advantage of allowing multiple genetic constructs to be introduced into cells simultaneously, each targeting a different part of the genome.
CRISPR/Cas9 technology for animal transgenesis finds application in conjunction with microinjection and SCNT techniques. In several reports, it was described that primary goat fibroblasts could be successfully modified at 80–90% efficiency
[29][50][51]. Fibroblasts with a diallel knock-out of the myostatin gene were used for SCNT, resulting in viable transgenic offspring
[52]. One of the proposed ways to eliminate genetic mosaicism is the direct injection of the CRISPR/Cas9 system components into metaphase II oocytes or early zygotes. Alternatively, electroporation of zygotes with the Cas9-RNP completely eliminates mosaicism
[53]. In sheep and cattle, injection of CRISPR/Cas9 into zygotes reduces mosaicism more effectively than injection of metaphase II oocytes
[2][54]. At the moment, fetal fibroblast-derived genetic modifications have been created for almost all types of farm animals, including cattle, pigs and goats, using the CRISPR/Cas9 system
[29][52][55][56].
The CRISPR/Cas9 system is widely used in many areas of livestock transgenesis. For instance, this system was successfully applied to a long-standing problem of sex selection in farm animals. Previously, various methods have been used to approach this issue: non-radioactive hybridization, fluorescence in situ hybridization, sex chromosome-based PCR and labeled Y-chromosome-specific probes
[2]. By knocking in the
eGFP in the Y chromosome of an embryonic bovine fibroblast (bovine fetal fibroblast (BFF)) system and subsequently transferring the resulting Y-Chr-eGFP construct using the SCNT method, it was possible to determine XY embryos by simply tracing a color label
[57].
One of the first genes to be targeted using this technique was
MSTN, which regulates muscle mass. In 2014–2015, researchers were able to create mutant sheep with higher body weight and muscle mass than their wild-type counterparts by editing this gene using the CRISPR/Cas9 system
[58][59]. These initial experiments paved the way for further application of genome editing in small ruminants.
Subsequent studies have demonstrated the versatility of the CRISPR/Cas9 system for multiplex gene editing in sheep. For example, efficient editing of three genes—
MSTN,
ASIP (agouti-signaling protein) and
BCO2 (β-carotene oxygenase 2)—was achieved using this method
[60][61]. Importantly, these studies showed minimal off-target effects. This could be particularly valuable for generating new lines of farm animals with multiple desired traits, as many economically important features are controlled by multiple loci.
In addition to gene knock-outs, the CRISPR/Cas9 system can also be used to generate animals with specific point mutations. For instance, Zhou et al. reported a high efficiency of single nucleotide substitution in the
SOCS2 gene, which controls body weight, size and milk production in sheep
[62]. These findings highlight the tremendous potential of the CRISPR/Cas9 system for creating GM farm animals with improved productivity and economic value.
Genetic editing methods have been mainly used in mammalian species of farm animals, while birds, which are equally important for agriculture and also serve as model organisms in biology, have not yet been a focus of genetic manipulation efforts. However, the chicken is one of the most widely farmed bird species and has been instrumental in several scientific breakthroughs. For instance, the first cholera vaccine developed by Louis Pasteur was tested in 1878 on domestic chickens. Additionally, the chicken was the first non-mammalian species to have its genome sequenced, and comparative analysis of the chicken genome has aided in the discovery of new genes and their functions in both animals and humans
[63].
Therefore, using the CRISPR/Cas9 system to produce GM chickens with desirable traits could be highly beneficial for agricultural and industrial applications. However, adapting the genetic editing tools that are commonly used in mammals to birds poses significant challenges due to differences in development and physiology. In particular, accessing and manipulating the fertilized oocyte nucleus in birds presents technical difficulties
[64][65].
Despite multiple challenges, scientists have successfully implemented the CRISPR/Cas9 system in avian species. For instance, Koslová et al. were able to produce chickens that are resistant to avian leukosis virus (ALV)
[66]. Another notable example is the generation of
MSTN knock-out chickens, wherein nickase D10A-Cas9 (Cas9n), a mutant Cas9 protein, was employed to minimize any non-specific double-strand breaks and off-target effects
[67]. In mammals, mutations in the
MSTN gene result in a distinct “double” musculature phenotype due to accelerated muscle growth. Similarly,
MSTN knock-out chickens and quails exhibit significantly larger skeletal muscles
[67][68][69][70]. Furthermore, Park et al. were able to create
G0S2 (G0/G1 switch gene 2) gene-edited chickens utilizing the CRISPR/Cas9 system
[71].
G0S2 serves as an inhibitor of adipose triglyceride lipase (ATGL, also known as protein 2 containing a patatin-like phospholipase domain), which catalyzes the first step of lipolysis (hydrolysis of triacylglycerols to diacylglycerols). The resulting
G0S2 knock-out chickens can be utilized as model animals in studies on obesity, providing an alternative to rodents
[72].
Recent advances in multiplex gene editing, single nucleotide substitution and the application of CRISPR/Cas9 technology to birds demonstrate its great potential for agricultural and industrial applications, while also providing new animal models for human and animal research.