The used by several groups to avoid the problem

The emergence of techniques like mapping, sequencing, and analyzing DNA information at genomic level adds a new dimension to animal breeding. Advances in genotyping and sequencing have improved our capacity to explore livestock genomes that further our comprehension of genomic selection. Recent advances in gene editing allows geneticists to introduce any common characteristic into any breed has the capability of improving animal genetics for meeting increasing agricultural and biomedical needs with less environmental impact (Tan et al., 2012, 2013). Genome editing in large animals has tremendous practical applications, from more precise models for medical research through improved animal welfare and production efficiency. Fish was first gene edited large animal of potential commercial importance (Zhu et al., 1986; Hackett and Alvarez, 2000; Devlin et al., 2009). From then, several lines of transgenic livestock have been engineered for producing valuable biomedical medicines and for agriculture (Tan et al., 2012).

The pronuclear microinjection (MI) of exogenous DNA directly into one-cell-stage embryos was the initial technique used to produce a transgenic animal (Gordon et al., 1980). By this technique the first genetically engineered farm animal was produced over 30 years ago (Hammer et al., 1985). Pronuclear injection has numerous practical limitations. As the integration is random and the constructs are usually integrated in multiple copies as concatemers, it was difficult to control the expression level. After that, successful somatic cell nuclear transfer (SCNT) using an embryo-derived differentiated cell population resulted in the breakthrough for genetically engineered animals, Dolly the cloned sheep in 1996 (Campbell et al., 1996). SCNT has since then been used to produce cloned cattle, goats and pigs (Laible et al., 2015; Polejaeva et al., 2015; Tan et al., 2016). Other approach successfully used by several groups to avoid the problem with low transgene integration efficiency was viral mediated gene transfer. Integrating viruses retain all of the problems of random integration associated with pronuclear injection, and because of their smaller cargo size, the problems with promoter strength and specificity are usually worse. In addition, because of the viral elements included, progressive silencing over time worsened with viral integration methods (Hofmann et al., 2006).

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The fundamentally novel innovation that has made the approaching revolution in gene editing possible is the ability to definitely target particular areas of the genome. This technology removes basically the greater part of the issues related with transgenic animals, because native promoter elements and splicing can be used for correct gene regulation, and the variability and gene silencing associated with random integration is eliminated. Instead of a largely random effect, gene editing can now be well controlled. The three major technologies for introducing site-specific double-stranded DNA breaks into genomes; a) zinc finger nucleases, ZFNs, b) transcription activator-like element nucleases, TALENs, and c) lustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) (Gaj et al., 2013; Kim and Kim, 2014) are effective in livestock genomes (Carlson et al., 2012; Tan et al., 2013). These DNA nucleases mediate targeted genetic alterations by enhancing the DNA mutation rate via induction of double-strand breaks at a predetermined genomic site. Contrasted with traditional homologous recombination-based gene targeting, DNA nucleases, also referred to as Genome Editors (GEs), can increase the targeting rate around 10,000-to 100,000-fold. Rather than all other DNA nucleases, that depend on protein– DNA official, CRISPR/Cas9 utilizes RNA to build up a specific binding of its DNA nuclease. Besides its capability to facilitate multiplexed genomic modifications in one shot, the CRISPR/Cas is much easier to design compared to all other DNA nucleases. Current results indicate that any DNA nuclease can be successfully employed in a broad range of organisms which renders them useful for improving the understanding of complex physiological systems such as reproduction, producing transgenic animals, including creating large animal models for human diseases, creating specific cell lines, and plants, and even for treating human genetic diseases.

Among some examples, cows and goats can be used as bioreactors to produce certain substances in their milk and pigs can be used as organ donors. A list of gene edited livestock for the different purposes is shown in Table 1.

Application of recent gene editing technologies in livestock includes;

a) Disease resistance

Recently, disease resistance could be demonstrated in cattle that became resistant against an infection with M. bovis with the aid of DNA nuclease-mediated genetic modifications (Gao et al., 2017; Wu et al.,2015). Researchers employed Cas9 nickase to introduce the NRAMP1 gene (natural resistance to infection with intracellular pathogens 1) into the bovine genome. The inserted NRAMP1 was correctly expressed and provided cattle with increased resistance to infection with M. bovis, which is the mycobacterial pathogen that causes bovine tuberculosis (Gao et al., 2017). Other prominent example is the production of pigs resistant against infection with the Porcine Reproductive and Respiratory Syndrome (PRRS) Virus (PRRSV) via genetic knockout of the CD163 receptor. The CD163-KO animals were completely protected against all symptoms of the PRRS infection with a single PRRSV isolate (Whitworth et al., 2016). The RELA gene was edited to cause resistance to the African swine fever, a viral infection that affects domestic pigs. After three days in vitro, 21 % of the embryos screened positive for edits in the RELA locus, and five live piglets were born which carried the porcine RELA mutation (Lillico et al., 2013). Liu and coworkers demonstrated mastitis resistance in cows by targeting human lysozyme gene to ?-casein locus using ZFN technique (Liu et al., 2014).

b) Increased productivity

The most prominent example relates to the genetic knockout of the myostatin gene (MSTN). MSTN is a negative regulator of the growth hormone family and thus limits skeletal muscular growth. The MSTN knockout leads to enhanced formation of skeletal muscles which could be beneficial for meat production under certain conditions. With the aid of DNA nucleases, the MSTN-KO phenotype was successfully induced in cattle, pigs, sheep and goats (Bi et al., 2016; Guo et al., 2016; Ni et al., 2014; Proudfoot et al., 2015; Wang et al., 2015; Yu et al., 2016).

c) Nutritional quality of milk

ZFN method was used to knocked out the sblg gene.which encode the bovine whey protein ?-lactoglobulin, a major milk protein and a dominant allergen. This method was successfully demonstrated in cattle and goat (Yu et al., 2011; Cui et al., 2015; Ni et al., 2014; Song et al., 2015; Xiong et al., 2013). The modified animal produced milk with a significantly altered protein composition, having elevated casein levels, but lacking any ?-lactoglobulin which constitutes the main allergenic component in bovine milk.

d) Production of allergen-reduced or allergen-free animal-derived products

Using CRISPR/Cas, the genes encoding for ovalbumin and ovomucoid have been knocked out in an effort to remove the two major allergenic components from egg white. This could render eggs digestible for wider range of consumers that could otherwise not consume chicken eggs (Oishi, Yoshii, Miyahara, Kagami, & Tagami, 2016).

e) Biomedicine

Several studies have already reported the production of genetically modified pigs for various biomedical purposes with the aid of DNA nucleases. The genetic knockout of a number of genes, including ?1,3-galactosyltransferase (GGTA1-gene), that encodes a sugar epitope on the surface of porcine cells and plays a major role in xenotransplantation (Butler et al., 2016; Hauschild et al., 2011; Petersen et al., 2016), the knockout of genes coding for PPAR-? (peroxisome proliferator-activated Receptor Gamma) and LDL (Low density lipoprotein) to produce large animal models for cardiovascular diseases (Carlson et al., 2012; Yang et al., 2011), DMD (Duchenne Muscular Disease) to produce a model for genetically induced muscular dystrophy (Carlson et al., 2012; Klymiuk et al., 2013), APC (Adenomatous-polyposis-coli Protein) to generate a model for certain types of intestinal cancer (Flisikowska et al., 2012; Tan et al., 2013) and the knockout of the gene coding for von Willebrand factor (vWF) to create a model for coagulation disorders (Hai, Teng, Guo, Li, & Zhou, 2014).

f) Animal welfare

The polled (hornless) trait is a desired trait in farmed cattle breed. Horned cattle can injure other animals in the herds or people working with the animals. Dehorning of cattle is associated with pain and stress for the animal and is also a costly procedure. Using TALENs, Carlson et al. achieved to introgress the causative celtic mutation (Pc) into the Holstein cattle genome resulting in a polled phenotype of the offspring (Carlson et al., 2016).


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