Submitted Successfully!
To reward your contribution, here is a gift for you: A free trial for our video production service.
Thank you for your contribution! You can also upload a video entry or images related to this topic.
Version Summary Created by Modification Content Size Created at Operation
1 -- 2870 2023-08-21 11:32:04 |
2 layout Meta information modification 2870 2023-08-22 02:30:25 |

Video Upload Options

Do you have a full video?


Are you sure to Delete?
If you have any further questions, please contact Encyclopedia Editorial Office.
Popova, J.; Bets, V.; Kozhevnikova, E. Precision Genome-Editing Tools Successfully Used in Livestock. Encyclopedia. Available online: (accessed on 20 June 2024).
Popova J, Bets V, Kozhevnikova E. Precision Genome-Editing Tools Successfully Used in Livestock. Encyclopedia. Available at: Accessed June 20, 2024.
Popova, Julia, Victoria Bets, Elena Kozhevnikova. "Precision Genome-Editing Tools Successfully Used in Livestock" Encyclopedia, (accessed June 20, 2024).
Popova, J., Bets, V., & Kozhevnikova, E. (2023, August 21). Precision Genome-Editing Tools Successfully Used in Livestock. In Encyclopedia.
Popova, Julia, et al. "Precision Genome-Editing Tools Successfully Used in Livestock." Encyclopedia. Web. 21 August, 2023.
Precision Genome-Editing Tools Successfully Used in Livestock

Genome editing of farm animals has undeniable practical applications. It helps to improve production traits, enhances the economic value of livestock, and increases disease resistance. Gene-modified animals are also used for biomedical research and drug production and demonstrate the potential to be used as xenograft donors for humans.

genome editing livestock CRISPR/Cas9

1. Introduction

More than 10 thousand years have passed since the domestication of various animal species by humans. Since then, much has changed in approaches to animal breeding. The modern agricultural industry is developing at an intensive pace and setting new challenges like the safety of breeding technologies and reducing the impact on the environment [1][2]. The traditional approach to livestock and domestic animal breeding relies on the positive and negative selection of the desired characteristics and usually takes generations to select and stabilize genetic traits in a population. In contrast, the relatively recent technologies of genome editing offer a one-step generation of an animal with predefined genetic and phenotypic features based on the knowledge of gene functions [3]. The term “GMO” (genetically modified organism) is used to refer to an organism whose genome has been deliberately altered in any way, including alterations that can occur naturally [4]. To date, the science of creating transgenic animals is one of the fastest-growing biotechnology fields, as it promises many advantages in a short period of time. However, even though genome engineering is speedy and often offers precise control over the traits, it raises strong safety concerns regarding the expansion and use of genetic modifications in contrast to traditional breeding, which is envisioned as safe [5][6].
Genetic modification of laboratory rodents was commonly implemented more than 40 years back, which soon led to the first attempts to transfer this technology to farm animals [7][8]. As a result, applied genetic engineering in breeding and biotechnology also underwent rapid development [9]. Despite all challenges, genetically modified (GM) farm animals have been produced, including cattle, sheep, goats, pigs, rabbits, chickens and fish [2].
Research in the field of gene engineering of farm animals has mainly focused on improving the efficiency of food production and animal health and welfare. Attempts are also made at reducing the impact of livestock products on human health and the environment [10][11]. In medical research, gene editing of farm animals can be used in a wide range of applications, from large-scale protein expression to the creation of humanized organs for transplantation (xenografts) [12][13][14]. Some large animals can be successfully used as preclinical models in testing drugs, artificial implants or surgical procedures [15][16]. The major strategy for agricultural GM clones is the maintenance of breeding stocks, and not productive stocks per se. Such breeding animals allow the creation of highly effective healthy herds by increasing the number of breeders with the desired traits. Subsequently, these animals are used for conventional breeding, and the offspring obtained from them can be used in production [17].
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].
Not long ago, researchers introduced a mouse gene that regulates body temperature into a pig. This improvement allowed the pigs to maintain a physiologically optimal temperature in cold weather by burning fat, which resulted in 24% less adipose tissue than in normal pigs [24]. This genomic change offers the production of pork with a low fat content, which may be considered beneficial to humans. At the same time, such transgenes are less demanding in terms of temperature maintenance in a facility, affording refined economic efficiency of such GM animals [24]. Other essential needs in livestock maintenance are successfully addressed by means of genome engineering. For instance, transgenic goats bearing knock-out mutations in two genes that inhibit hair and muscle growth allow breeders to produce more cashmere and meat [25].
Targeted mutagenesis has also been applied to the genes encoding ovalbumin and ovomucoid, common egg allergens in chickens, using the clusters of regularly interspaced short palindromic repeats (CRISPR) method. The researchers hope to apply this technique in producing hypoallergenic eggs in the future [26].
Sheep and pigs are actively used for genome editing in order to recapitulate key features of human diseases. Williams et al. demonstrate a sheep model that reproduces human hypophosphatasia, a rare metabolic bone disease, using CRISPR/Cas9 (CRISPR-associated nuclease 9) as a molecular tool [27]. Likewise, large animals with the NUP155 knock-out mutation are useful models in heart tissue physiology research [28][29][30]. Fan et al. reported the creation of IFNAR knock-out sheep using CRISPR/Cas9 in combination with somatic cell nuclear transfer (SCNT) to produce a large animal model with high susceptibility to Zika virus [31].
It is also worth mentioning a study that created a model with impaired pancreatic development in sheep. The group of scientists applied CRISPR/Cas9 to PDX1 (pancreatic and duodenal homeobox protein 1), which is essential to pancreatic growth [32][33]. CRISPR/Cas9 combined with a direct oocyte microinjection was used to disrupt PDX1, resulting in homozygous mutant fetuses that were pancreas-free. The results of these promising efforts highlight the potential of gene-edited sheep as hosts for human xenograft growth via blastocyst complementation [32][33].

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.


  1. Van Eenennaam, A.L.; Young, A.E. Genetic Improvement of Food Animals: Past and Future. In Encyclopedia of Food Security and Sustainability; Elsevier: Amsterdam, The Netherlands, 2018; pp. 171–180.
  2. Singh, P.; Ali, S.A. Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals. Vet. Sci. 2021, 8, 122.
  3. McFarlane, G.R.; Salvesen, H.A.; Sternberg, A.; Lillico, S.G. On-Farm Livestock Genome Editing Using Cutting Edge Reproductive Technologies. Front. Sustain. Food Syst. 2019, 3, 106.
  4. Turnbull, C.; Lillemo, M.; Hvoslef-Eide, T.A.K. Global Regulation of Genetically Modified Crops Amid the Gene Edited Crop Boom—A Review. Front. Plant Sci. 2021, 12, 630396.
  5. Prakash, D.; Verma, S.; Bhatia, R.; Tiwary, B.N. Risks and Precautions of Genetically Modified Organisms. ISRN Ecol. 2011, 2011, 369573.
  6. Hallerman, E.M.; Bredlau, J.P.; Camargo, L.S.A.; Dagli, M.L.Z.; Karembu, M.; Ngure, G.; Romero-Aldemita, R.; Rocha-Salavarrieta, P.J.; Tizard, M.; Walton, M.; et al. Towards progressive regulatory approaches for agricultural applications of animal biotechnology. Transgenic Res. 2022, 31, 167–199.
  7. Wells, D.J. Genetically Modified Animals and Pharmacological Research. Comp. Vet. Pharmacol. 2010, 199, 213–226.
  8. Niemann, H.; Kues, W.A. Transgenic farm animals: An update. Reprod. Fertil. Dev. 2007, 19, 762–770.
  9. Eriksson, S.; Jonas, E.; Rydhmer, L.; Röcklinsberg, H. Invited review: Breeding and ethical perspectives on genetically modified and genome edited cattle. J. Dairy Sci. 2018, 101, 1–17.
  10. Van Eenennaam, A.L. Application of genome editing in farm animals: Cattle. Transgenic Res. 2019, 28, 93–100.
  11. Niemann, H.; Kues, W.; Carnwath, J.W. Transgenic Farm Animals: Current Status and Perspectives for Agriculture and Biomedicine; Springer: Berlin/Heidelberg, Germany, 2009.
  12. Houdebine, L.-M. Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 2009, 32, 107–121.
  13. Hata, T.; Uemoto, S.; Kobayashi, E. Transplantable liver production plan. Organogenesis 2013, 9, 235–238.
  14. Hryhorowicz, M.; Zeyland, J.; Słomski, R.; Lipiński, D. Genetically Modified Pigs as Organ Donors for Xenotransplantation. Mol. Biotechnol. 2017, 59, 435–444.
  15. West, J.; Gill, W.W. Genome Editing in Large Animals. J. Equine Vet. Sci. 2016, 41, 1–6.
  16. Kubo, Y.; Yamashita, K.; Saito, T.; Tanaka, K.; Makino, T.; Takahashi, T.; Kurokawa, Y.; Yamasaki, M.; Eguchi, H.; Doki, Y.; et al. Heparinized swine models for better surgical/endoscopic training. DEN Open 2021, 2, e64.
  17. Tamme, R.; Laing, D.; Steinmann, W.-D.; Bauer, T. Transgenic Livestock for Food Production, Introduction. In Encyclopedia of Sustainability Science and Technology; Elsevier: Amsterdam, The Netherlands, 2012; pp. 10812–10814.
  18. Keiser, N.W.; Engelhardt, J.F. New animal models of cystic fibrosis: What are they teaching us? Curr. Opin. Pulm. Med. 2011, 17, 478–483.
  19. Noble, M.S.; Rodriguez-Zas, S.; Cook, J.B.; Bleck, G.T.; Hurley, W.L.; Wheeler, M.B. Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk. J. Anim. Sci. 2002, 80, 1090–1096.
  20. Carlson, D.F.; Lancto, C.A.; Zang, B.; Kim, E.-S.; Walton, M.; Oldeschulte, D.; Seabury, C.; Sonstegard, T.S.; Fahrenkrug, S.C. Production of hornless dairy cattle from genome-edited cell lines. Nat. Biotechnol. 2016, 34, 479–481.
  21. Young, A.E.; Mansour, T.A.; McNabb, B.R.; Owen, J.R.; Trott, J.F.; Brown, C.T.; Van Eenennaam, A.L. Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull. Nat. Biotechnol. 2019, 38, 225–232.
  22. Niu, Y.; Zhao, X.; Zhou, J.; Li, Y.; Huang, Y.; Cai, B.; Liu, Y.; Ding, Q.; Zhou, S.; Zhao, J.; et al. Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9. Reprod. Fertil. Dev. 2018, 30, 307.
  23. Fabre, S.; Pierre, A.; Mulsant, P.; Bodin, L.; Di Pasquale, E.; Persani, L.; Monget, P.; Monniaux, D. Regulation of ovulation rate in mammals: Contribution of sheep genetic models. Reprod. Biol. Endocrinol. 2006, 4, 20.
  24. Zheng, Q.; Lin, J.; Huang, J.; Zhang, H.; Zhang, R.; Zhang, X.; Cao, C.; Hambly, C.; Qin, G.; Yao, J.; et al. Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proc. Natl. Acad. Sci. USA 2017, 114, E9474–E9482.
  25. Wang, X.; Yu, H.; Lei, A.; Zhou, J.; Zeng, W.; Zhu, H.; Dong, Z.; Niu, Y.; Shi, B.; Cai, B.; et al. Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system. Sci. Rep. 2015, 5, srep13878.
  26. Oishi, I.; Yoshii, K.; Miyahara, D.; Kagami, H.; Tagami, T. Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci. Rep. 2016, 6, 23980.
  27. Williams, D.K.; Pinzón, C.; Huggins, S.; Pryor, J.H.; Falck, A.; Herman, F.; Oldeschulte, J.; Chavez, M.B.; Foster, B.L.; White, S.H.; et al. Genetic engineering a large animal model of human hypophosphatasia in sheep. Sci. Rep. 2018, 8, 16945.
  28. Hu, S.; Wang, Z.; Polejaeva, I. 40 KNOCKOUT OF GOAT NUCLEOPORIN 155 (NUP155) GENE USING CRISPR/Cas9 SYSTEMS. Reprod. Fertil. Dev. 2014, 26, 134.
  29. Ni, W.; Qiao, J.; Hu, S.; Zhao, X.; Regouski, M.; Yang, M.; Polejaeva, I.A.; Chen, C. Efficient Gene Knockout in Goats Using CRISPR/Cas9 System. PLoS ONE 2014, 9, e106718.
  30. Program and Abstracts of the 14th Transgenic Technology Meeting (TT2017): Snowbird Resort, Salt Lake City, Utah, USA, 1–4 October 2017. Transgenic Res. 2017, 26, 1–45.
  31. Fan, Z.; Yang, M.; Regouski, M.; Polejaeva, I.A. Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer. Methods Mol. Biol. 2019, 1874, 373–390.
  32. Vilarino, M.; Rashid, S.T.; Suchy, F.P.; McNabb, B.R.; van der Meulen, T.; Fine, E.J.; Ahsan, S.D.; Mursaliyev, N.; Sebastiano, V.; Diab, S.S.; et al. CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci. Rep. 2017, 7, 17472.
  33. Vilarino, M.; Suchy, F.P.; Rashid, S.T.; Lindsay, H.; Reyes, J.; McNabb, B.R.; van der Meulen, T.; Huising, M.O.; Nakauchi, H.; Ross, P.J. Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic Res. 2018, 27, 525–537.
  34. Carroll, D. Genome editing: Past, present, and future. Yale J. Biol. Med. 2017, 90, 653–659.
  35. Sun, Z.; Wang, M.; Han, S.; Ma, S.; Zou, Z.; Ding, F.; Li, X.; Li, L.; Tang, B.; Wang, H.; et al. Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA. Sci. Rep. 2018, 8, 15430.
  36. Hauschild, J.; Petersen, B.; Santiago, Y.; Queisser, A.-L.; Carnwath, J.W.; Lucas-Hahn, A.; Zhang, L.; Meng, X.; Gregory, P.D.; Schwinzer, R.; et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 2011, 108, 12013–12017.
  37. Liu, X.; Wang, Y.; Tian, Y.; Yu, Y.; Gao, M.; Hu, G.; Su, F.; Pan, S.; Luo, Y.; Guo, Z.; et al. Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases. Proc. R. Soc. B Boil. Sci. 2014, 281, 20133368.
  38. Flisikowska, T.; Thorey, I.S.; Offner, S.; Ros, F.; Lifke, V.; Zeitler, B.; Rottmann, O.; Vincent, A.; Zhang, L.; Jenkins, S.; et al. Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases. PLoS ONE 2011, 6, e21045.
  39. Geurts, A.M.; Cost, G.J.; Freyvert, Y.; Zeitler, B.; Miller, J.C.; Choi, V.M.; Jenkins, S.S.; Wood, A.; Cui, X.; Meng, X.; et al. Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases. Science 2009, 325, 433.
  40. Lillico, S.G.; Proudfoot, C.; King, T.J.; Tan, W.; Zhang, L.; Mardjuki, R.; Paschon, D.E.; Rebar, E.J.; Urnov, F.D.; Mileham, A.J.; et al. Mammalian interspecies substitution of immune modulatory alleles by genome editing. Sci. Rep. 2016, 6, 21645.
  41. Bedell, V.M.; Wang, Y.; Campbell, J.M.; Poshusta, T.L.; Starker, C.G.; Krug, R.G., 2nd; Tan, W.; Penheiter, S.G.; Ma, A.C.; Leung, A.Y.H.; et al. In vivo genome editing using a high-efficiency TALEN system. Nature 2012, 491, 114–118.
  42. Sanjana, N.E.; Cong, L.; Zhou, Y.; Cunniff, M.M.; Feng, G.; Zhang, F. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 2012, 7, 171–192.
  43. Moscou, M.J.; Bogdanove, A.J. A Simple Cipher Governs DNA Recognition by TAL Effectors. Science 2009, 326, 1501.
  44. Method of the Year 2011. Nat. Methods 2011, 9, 1.
  45. Moghaddassi, S.; Eyestone, W.; Bishop, C.E. TALEN-Mediated Modification of the Bovine Genome for Large-Scale Production of Human Serum Albumin. PLoS ONE 2014, 9, e89631.
  46. Carlson, D.F.; Tan, W.; Lillico, S.G.; Stverakova, D.; Proudfoot, C.; Christian, M.; Voytas, D.F.; Long, C.R.; Whitelaw, C.B.A.; Fahrenkrug, S.C. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. USA 2012, 109, 17382–17387.
  47. Cong, L.; Ran, F.A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.D.; Wu, X.; Jiang, W.; Marraffini, L.A.; et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339, 819–823.
  48. Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821.
  49. Mali, P.; Esvelt, K.M.; Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 2013, 10, 957–963.
  50. Hao, F.; Yan, W.; Li, X.; Wang, H.; Wang, Y.; Hu, X.; Liu, X.; Liang, H.; Liu, D. Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing. Int. J. Biol. Sci. 2018, 14, 427–436.
  51. Kalds, P.; Zhou, S.; Cai, B.; Liu, J.; Wang, Y.; Petersen, B.; Sonstegard, T.; Wang, X.; Chen, Y. Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era. Front. Genet. 2019, 10, 750.
  52. Zinovieva, N.A.; Volkova, N.A.; Bagirov, V.A.; Brem, G. Transgenic farm animals: Status of the current researches and the future. Ecol. Genet. 2015, 13, 58–76.
  53. Hashimoto, M.; Yamashita, Y.; Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 2016, 418, 1–9.
  54. O’Neil, E.V.; Brooks, K.; Burns, G.W.; Ortega, M.S.; Denicol, A.C.; Aguiar, L.H.; Pedroza, G.H.; Benne, J.; Spencer, T.E. Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep. Mol. Reprod. Dev. 2020, 87, 142–151.
  55. Heo, Y.T.; Quan, X.; Xu, Y.N.; Baek, S.; Choi, H.; Kim, N.-H.; Kim, J. CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells. Stem Cells Dev. 2015, 24, 393–402.
  56. Tan, W.; Carlson, D.F.; Lancto, C.A.; Garbe, J.R.; Webster, D.A.; Hackett, P.B.; Fahrenkrug, S.C. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc. Natl. Acad. Sci. USA 2013, 110, 16526–16531.
  57. Zhao, X.; Nie, J.; Tang, Y.; He, W.; Xiao, K.; Pang, C.; Liang, X.; Lu, Y.; Zhang, M. Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration. Front. Vet. Sci. 2020, 7, 199.
  58. Hongbing, H.; Yonghe, M.; Tao, W.; Ling, L.; Xiuzhi, T.; Rui, H.; Shoulong, D.; Kongpan, L.; Feng, W.; Ning, L.; et al. One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system. Front. Agric. Sci. Eng. 2014, 1, 2–5.
  59. Crispo, M.; Mulet, A.P.; Tesson, L.; Barrera, N.; Cuadro, F.; dos Santos-Neto, P.C.; Nguyen, T.H.; Crénéguy, A.; Brusselle, L.; Anegón, I.; et al. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PLoS ONE 2015, 10, e0136690.
  60. Wang, X.; Niu, Y.; Zhou, J.; Yu, H.; Kou, Q.; Lei, A.; Zhao, X.; Yan, H.; Cai, B.; Shen, Q.; et al. Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep. Sci. Rep. 2016, 6, 32271.
  61. Wang, X.; Liu, J.; Niu, Y.; Li, Y.; Zhou, S.; Li, C.; Ma, B.; Kou, Q.; Petersen, B.; Sonstegard, T.; et al. Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep. BMC Genom. 2018, 19, 397.
  62. Zhou, S.; Cai, B.; He, C.; Wang, Y.; Ding, Q.; Liu, J.; Liu, Y.; Ding, Y.; Zhao, X.; Li, G.; et al. Programmable Base Editing of the Sheep Genome Revealed No Genome-Wide Off-Target Mutations. Front. Genet. 2019, 10, 215.
  63. Stern, C. The chick model system: A distinguished past and a great future. Int. J. Dev. Biol. 2018, 62, 1–4.
  64. Park, T.S.; Lee, H.J.; Kim, K.H.; Kim, J.-S.; Han, J.Y. Targeted gene knockout in chickens mediated by TALENs. Proc. Natl. Acad. Sci. USA 2014, 111, 12716–12721.
  65. Park, T.S.; Han, J.Y. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc. Natl. Acad. Sci. USA 2012, 109, 9337–9341.
  66. Koslová, A.; Kučerová, D.; Reinišová, M.; Geryk, J.; Trefil, P.; Hejnar, J. Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells. Viruses 2018, 10, 605.
  67. Kim, G.-D.; Lee, J.H.; Song, S.; Kim, S.W.; Han, J.S.; Shin, S.P.; Park, B.-C.; Park, T.S. Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase. FASEB J. 2020, 34, 5688–5696.
  68. Lee, J.; Kim, D.-H.; Lee, K. Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide. Int. J. Mol. Sci. 2020, 21, 1504.
  69. Lee, J.; Ma, J.; Lee, K. Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail. Proc. Natl. Acad. Sci. USA 2019, 116, 13288–13292.
  70. Lee, J.; Kim, D.-H.; Karolak, M.C.; Shin, S.; Lee, K. Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing. Proc. Natl. Acad. Sci. USA 2022, 119, e2214344119.
  71. Park, T.S.; Park, J.; Lee, J.H.; Park, J.-W.; Park, B.-C. Disruption of G0/G1 switch gene 2 ( G0S2 ) reduced abdominal fat deposition and altered fatty acid composition in chicken. FASEB J. 2018, 33, 1188–1198.
  72. Park, T.S. —Invited Review—Gene-editing techniques and their applications in livestock and beyond. Anim. Biosci. 2023, 36, 333–338.
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to : , ,
View Times: 167
Revisions: 2 times (View History)
Update Date: 22 Aug 2023
Video Production Service