Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications
Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3 billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as “dual purpose” applications.
Genetic modification of livestock genomes by humans is not a novel concept. From the dawn of civilization, humans have engaged in the practice of deliberately modifying the animal genomes by selectively breeding them. In fact, domestication of all animals has its roots in selective breeding, where animals of perceived “high merit” were selectively bred for improving docility and adaptation to human management, then further selected for breed type and conformation, high milk production, carcass weight, disease resistance, adaptations to local environment, and other traits deemed critical for their respective applications. These techniques greatly improved the rate of genetic progress for a large suite of economically important traits and continue to evolve today. The advent of genomic technologies including marker arrays, have added to the power of traditional animal breeding technologies and are aiding in the rate of genetic progress. Unfortunately, these technologies are not amenable for the manipulation or isolation of specific novel traits or alleles that may be of singular interest to the animal breeder or researchers. Introgression of novel alleles, transfer of alleles between breeds, and introduction of exogenous traits require sophisticated genetic modifications. The latter is especially important for the use of animals in biomedical studies, where targeted genetic modifications are required in the animals to mimic human mutations or diseased states, or humanization of the genome for xenotransplantation studies. In this context, the genome editors are leading the way in livestock genetic engineering field. In this article, the current state of genetic engineering efforts in livestock will be discussed in the context of genome editors, and identify the many advantages they offer for agricultural and biomedical applications.
Genetic engineering in domestic animals: a progress report
In pioneering experiments by Szybalska and Szybalski about half a century ago, it was first demonstrated that exogenous genetic information can be transferred into mammalian cells by transforming cells deficient in hypoxanthine phosphoribosyl transferase (HPRT) to become resistant to HAT (for Hypothanthine, Aminopterin, and Thymidine) selection medium (Szybalska and Szybalski 1962). In the experiments that followed, exogenous DNA was microinjected into cultured cells (Capecchi 1980; Gordon et al. 1980; Graessmann et al. 1979), which set the stage for microinjection of DNA into pronucleus (PN) of mouse zygotes (Gordon et al. 1980) for the generation of first transgenic mice (Brinster et al. 1981). This was soon followed by the generation of first transgenic livestock species (Hammer et al. 1985). Though conceptually simple, the procedure in livestock is complicated by the fact that the zygote of livestock species is opaque and the PN are not readily visible. This together with the low efficiency of generating stable founders and long generation intervals and small number of offspring being born (in ruminants) makes it less than an ideal option for generating transgenic founders (Prather et al. 2008). Others have attempted precomplexing of the DNA with sperm and fertilized the oocytes for the delivery of exogenous DNA into the egg (Gandolfi 2000; Lavitrano et al. 2006, 1997). The process known as sperm mediated gene transfer (SMGT) suffers from a lack of reproducibility in generating transgenic animals (Brinster et al. 1989). Retroviruses that stably transduce intact zygotes and integrate into their genomes when placed into the perivitelline space have also been successfully utilized in generating transgenic farm animals (Cabot et al. 2001; Hofmann et al. 2003). Unlike PN injection or SMGT, the retroviral approach has been found to be more reliable in generating transgenic founders as the vectors actively integrate into the genome with very minimal viral sequence. The main draw backs with this approach are the limitations on the size of the transgene (~8 kb), and a lack of consistency in stable expression and transmission, even from the same lab. That said, this technique is still a standard for generating transgenic poultry (Federspiel and Hughes 1997; Solaiman et al. 2000). Another tool in the tool box for livestock geneticists are the mobile “jumping genes”, notably transposons. The transposons are naturally existing mobile elements that can stably integrate into the genomes with minimal recognition sequences. Additionally, by utilizing transposable elements that are not native to the host species, any concern of activating endogenous transposable elements is offset (Clark et al. 2007; Plasterk et al. 1999). Several transposable elements are currently in use for farm animal transgenesis (Carlson et al. 2011; Kong et al. 2008; Ni et al. 2008).
Gene targeting by homologous recombination
Barring the mode of delivery and mechanism of integration, all the above approaches suffer somewhat from the same set of limitations including the lack of reliable and reproducible integration at target sites. The apparent randomness of genomic integration brings with it a host of unwanted side effects such as lack of control over the copy number and consequently dosage of the transgene; aberrant expression in non-target tissues based on the site of integration or silencing of transgene (positional variegation); insertion into functional genes and mutating the endogenous genes (insertional mutagenesis); to name a few. This has necessitated a need for refined approaches that utilize homologous recombination (HR) mediated gene targeting and modification of the genome. HR is a mechanism whereby gene cassettes between isogenic/homologous sequences are exchanged (Capecchi 1989), resulting in a targeted gene knockin (KI) of transgenes or disruption of target genes with selection cassettes to generate knockout (KO) animals.
Promise of stem cells that weren’t to be
A major limitation of HR-based gene targeting in livestock is the lack of embryonic stem cells for any livestock species that are amenable to genetic modification and subsequent testing. The establishment of authentic embryonic stem cells (ESC) like the one in mouse remains elusive despite more than three decades of intensive investigations (Nowak-Imialek and Niemann 2012). Although progress has been made in establishing stem cells of embryonic origin (Alberio et al. 2010), the established lines are clearly distinct from the mouse ESC characterized by a naïve phenotype, and are similar to human ESC representing the “epiblast stem cell” phenotype. The stem cells are FGF-dependent instead of LIF-dependent, are flattened, and do not tolerate dispersal as single cells. Additionally, gene targeting and HR is inefficient in these epiblast-type stem cell types (Gafni et al. 2013). Induced pluripotent stem cells (iPSC) generated by upregulation of four reprogramming factors and cultured on LIF-based medium offer an alternative (Telugu et al. 2010, 2011). Contrary to the widely-touted advantages of stem cells, such as effective contribution to chimeras or in generating live offspring via NT, evidence in the livestock field so far has been underwhelming. Initial experiments with iPSC in NT experiments were less than encouraging, most likely due to persistent expression of reprogramming transgenes (Cuffee et al. 2014). It remains to be seen if the reported “naive”-type stem cell lines can contribute to chimeras and colonize the gonads (Telugu et al. 2010, 2011). In this regard, it would be prudent to actively pursue in parallel primordial germ cells (PGC), although germline specific imprinting could prove to be a barrier. Genetic modification utilizing PGC has been demonstrated in poultry (Macdonald et al. 2010). The PGC can be genetically modified in vitro and returned to chick embryos where they form eggs and sperm when the chicks reach sexual maturity. Conceivably, as an alternative to PGCs, spermatogonial stem cells (SSCs) of prized males could be genetically modified in culture and transplanted into surrogates lacking an endogenous germline for effective transmission of genetic modifications in months rather than over years as with conventional breeding. The feasibility of this approach has already been demonstrated in a proof of concept study in goats (Honaramooz et al. 2008). Goat SSCs, modified by AAV-transduction in vitro, were transplanted into the testes of recipients with depleted endogenous germ cells. Subsequent use of sperm from these bucks for in vitro fertilization readily produced transgenic embryos. Potentially, this could be accelerated even further by in vitro differentiation of pluripotent stem cells of embryonic ESC or somatic iPSCs into oocytes and sperm (Geijsen et al. 2004; Hubner et al. 2003). These so-called artificial gametes might then be used for fertilization to produce viable offspring (Hayashi et al. 2012, 2011). Progress in the stem cell field has been slow but tangible. The many advances in deriving and culturing stem cells provide a positive outlook for this otherwise nascent field.
Cloning is a reliable option
In the absence of robust stem cells, the somatic cell nuclear transfer (SCNT) or cloning has emerged as a method of choice for generating transgenic or gene targeted large animal models. Following successful demonstration that the nuclei of adult somatic cells could be reprogrammed to produce viable mammalian offspring (Wilmut et al. 1997), cloning from all domestic animals has now been reported (Cibelli et al. 1998; Onishi et al. 2000; Yin et al. 2005; Yong and Yuqiang 1998). The readers are referred to several excellent review articles on this subject, and will not be discussed in detail here (Galli et al. 2012; Whyte and Prather 2011). Briefly, the haploid genome of the oocytes arrested at the metaphase stage of meiosis II is removed alongside the extruded polar body. Following enucleation, the pre-screened somatic cell carrying the targeted genetic mutation is placed in the perivitelline space and fused by an electric pulse. The oocyte with its now reconstituted diploid genome will initiate the cleavage divisions and following transfer into recipients will generate live offspring. A requirement for pre-screening of the desired mutations in somatic cells (due to a relatively long generation interval), necessitated SCNT as a de facto option for generating genetically modified large animals. That said, SCNT is the most demanding of techniques for an average laboratory embarked on genetic engineering. The process is incredibly finicky, and is influenced by many factors including the species, source of donor cell, time of cell in culture, potential incomplete reprogramming of the epigenome of the donor nucleus in the reconstituted zygote, defects in extraembryonic membrane developments following implantation, among variables that effect outcome of this system.
At a cellular level, major hurdles remain in using fibroblasts or other somatic cells as a source material. Potential complications include the limited survival of fibroblasts in culture, and the poor efficiency of achieving the desired recombination events, typically ranging 1 in 106–107 in cultured cells, and time it takes to screen for the positive recombinants. In most cases, a round of gene targeting generates monoallelic modifications, requiring a second round of targeting for biallelic (homozygous) modifications that are a necessity to assess the altered/predicted phenotype. This typically entails another round of SCNT, embryo transfer, and harvesting of the fetal fibroblasts to perform a second gene targeting experiment. Given the already long gestation periods and length of time to reproductive age in large animals, generation of modified animals by nuclear transfer (cloning) and subsequent breeding to homozygosity, or performing multiple rounds of gene targeting and embryo transfer, both technically challenging and a cost prohibitive option.
Genetic engineering in the context of genome editors
The year 1996 marked two major events in genetic engineering arena, the advent of cloning (Campbell et al. 1996) and the first reports on the use of programmable genome editors (Kim et al. 1996). While, deservedly so, the cloning of dolly was received with much fan-fare, the response for genome editors has largely been muted. The genome editors, however, are beginning to capture public and scientific discourse alike in recent times. The editors, also referred to as site-specific nucleases or designer nucleases, are an innovative adaptation of the in vivo systems. They are either based of naturally occurring nucleases such as the Cas9 from the bacterial CRISPR/Cas system (Cong et al. 2013; Jinek et al. 2012, 2013; Mali et al. 2013) or the homing endonucleases identified from yeast gene-drives (Choulika et al. 1995); or, the artificial modular assemblies of zinc fingers (Kim et al. 1996) or the TALs (Christian et al. 2010) fused to a facultative nuclease such as Fok1. Albeit the differences, all editors perform the same basic function, which is to introduce a double strand break (DSB) at the target site. The introduction of DSB triggers either an error-prone non-homologous end joining (NHEJ) repair, or a conservative homology directed repair (HDR) pathway (Kass and Jasin 2010; Symington and Gautier 2011). The NHEJ events can be leveraged to introduce DSBs and subsequently errors within the open reading frame (ORF) resulting in out-of-frame mutations and knockout of the genes. When, the repair is facilitated by strand invasion into the repair template, the HDR pathway results in the introduction of subtle mutations or knockin of transgenes into the target site.
Among the four available editors, the CRISPR/Cas9 has emerged as a clear winner in the race for livestock genome editing. The system uses a 20 nucleotide RNA sequence as a guide along with a universal sequence (77 nucleotides) that permits loading into the Cas9 protein. The RNA is relatively easy to design and synthesize either in vitro, or purchased from many commercial vendors. Likewise, the Cas9 can be delivered as a DNA coding vector or in vitro transcribed RNA (both not preferable) or purchased as a commercial protein. The latter option in which the RNA is pre-complexed with the protein, and the ribonucleoprotein complex delivered into either cells for subsequent SCNT, or directly into the embryos. Already multiple groups including ours have demonstrated successful generation of knockout animals by injection of CRISPRs directly into the cytoplasm of zygotes (Chang et al. 2002; Chen et al. 2015; Hai et al. 2014; Kang et al. 2016; Park et al. 2017a; Peng et al. 2015; Petersen et al. 2016; Wang et al. 2015a, c, d; Whitworth et al. 2014; Zhou et al. 2016, 2015). Efforts have similarly been successful in our laboratory to generate knockin animals (Park et al. 2017b). The availability of on-the-shelf reagents, coupled with the ease of delivering the reagents including electroporation (Tanihara et al. 2016), and a high reliability of introducing designer edits is expected to fast track genome editing in livestock species. Conceptually, a typical reproductive biology laboratory with access to livestock animals, and an ability to harvest and handle embryos can generate genome edited animals. This is a far cry from the technically demanding and laborious SCNT or inefficient transgenic approaches of the past. If the recent spike in publications utilizing genome editors are any indication, this prediction is likely going to be true, and the true impact of CRISPRs (and other editors) will likely be clearly visible in the livestock genetic engineering field. From a SCNT perspective, although HR mediated gene targeting events are rare in somatic cells (1:106–107 cells), the efficiencies can be improved by greater than threefolds of magnitude by introducing a DSB (Rouet et al. 1994). This essentially eliminates a perceived bottleneck in SCNT, which is generating homozygous biallelic edits in one generation. Although the merits of embryo injections vs SCNT based approaches are hotly debated, and a clear winner has yet to emerge, it is clear that the livestock genetic engineering field is poised for a take-off. Combined with the sequencing of animal genomes, the animal biotechnology field is ready to realize the full potential in addressing global agricultural and biomedical challenges.
Next generation breeding technologies facilitated by genome editors to meet global agricultural challenges
By year 2050, the current 7 billion world population is projected to grow by another 2.6 billion (http://www.fao.org/fileadmin/templates/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf), with a concomitant 70% increase in the requirement for animal products. The increase in global population is expected to occur predominantly in developing countries, with the majority residing in urban areas. Majority of arable land is already under production, and the land use is being further impacted by urbanization, production of biofuels, and climate change. Secondly, the animal agriculture is continuously under public scanner with production practices related to animal welfare such as castration and dehorning occupying headlines. Finally, risks of global pandemics affecting animals, such as foot-and-mouth disease virus, or zoonotic diseases that affect both the humans and animals alike (e.g. influenza), are one-Health challenges that need to be tackled.
In the past five decades, even though major gains in animal agriculture have been achieved via the use of selective breeding and better management practices, sustained selection pressure on a singular production trait has created bottlenecks in agricultural systems, potentially due to linkage of traits. Additionally, introducing novel alleles or traits for creating a new phenotype in a population is painstakingly slow because of crossing over (meiotic recombination) during gametogenesis and subsequent mixing of the genomes following fertilization. At a minimum, 5–6 generations of backcrossing are required to introduce the desired phenotype into an existing breed. In cattle, it translates to 30 years (Smith 1989) for achieving 30% gain in genetic value. Consequently, new “next generation” animal breeding technologies are needed to enable animal breeders to take advantage of independent introduction and transmission of desirable traits.
Additionally, a better understanding of factors influencing farm animal health, reproduction, and nutrient utilization will no doubt benefit our ability to produce food animals, while comparative biological studies will shed light on similar concerns in human medicine (e.g. health, fertility, obesity). Representative examples where genetic engineering has already made an impact are below:
Modification of milk:
Production of hypoallergenic milk (Jabed et al. 2012).
Improving milk production. e.g. alpha-LA (lactalbumin) transgenic sow (Bleck et al. 1998)
‘Biofarming’: production of milk proteins and other proteins of pharmaceutical value e.g. recombinant human antithrombin produced in the milk of goats (marketed as ATryn) (Lavine 2009; Schmidt 2006) and recombinant human C1 esterase inhibitor produced in the milk of rabbits (marketed as Ruconest) (van Veen et al. 2012), human IL-2 in the milk of transgenic rabbits (Buhler et al. 1990), human tissue type plasminogen activator in goat milk (Ebert et al. 1991), active alpha-1-antitrypsin in sheep milk (Wright et al. 1991).
Modification of growth and carcass composition:
Fat-1 transgenic pig with elevated omega-3 fatty acid production- heart healthy pork (Lai et al. 2006)
Enhanced growth in porcine growth hormone transgenic pig (Vize 1988).
IGF-1 transgenic pigs with more loin mass and improved carcass characteristics (Pursel et al. 2004).
In addition, a myostatin (KO) pig and cattle that exhibit enhanced lean muscle mass throughout the carcass apparently due to muscle cell hyperplasia without any gross abnormalities-are currently being considered (Carlson et al. 2012; Wang et al. 2015b, d).
As can be expected there are many such genes that can be exploited for manipulating growth and carcass quality in meat producing animals.
Modification of disease resistance:
There are many aspects of disease resistance or susceptibility in livestock that are genetically determined. Other examples include the generation of prion-gene KO sheep (Denning et al. 2001), goats (Yu et al. 2006), and cattle (Kuroiwa et al. 2004) that are resistant for spongiform encephalopathies (Scrapie and BSE, respectively), and the lysostaphin-cow that resists staphylococcal mastitis (Wall et al. 2005). Deletion of specific genes/inclusion of synthetic constructs to confer disease resistance include influenza-free birds (Lyall et al. 2011). PRRS resistant pigs have been generated by targeted ablation of CD163 (Whitworth et al. 2016), while pigs resistant to African Swine Fever are generated by modification of NF-kB RelA subunit (Lillico et al. 2016). Likewise, cattle resistant to Bovine respiratory disease are being considered for modification. Several such avenues for disease resistance can be identified and disease resistant transgenic lines created. With the improvement of gene targeting efficiencies, it is worth revisiting previous models that have failed to express transgenes efficiently.
Modification of reproductive performance and prolificacy:
A prime example are mutations in oocyte-derived growth factors of the TGF-beta family and a related receptor. Mutations in growth differentiation factor 9, bone morphogenetic protein 15 and activin receptor-like kinase 6 are resulting in increased ovulation rates and thus litter size (McNatty et al. 2005). Several potential candidates have been identified which affect reproductive performance. One of these is the estrogen receptor gene. ESR gene is associated with increased litter size in pigs (1.4 more piglets between two homozygous genotypes) (Goliasova and Wolf 2004). Introduction of such genes can improve prolificacy in diverse breeds. Novel applications include germline ablation for germ cell transplantation (surrogate sires) for accelerating genetic selection (Park et al. 2017a).
Generation of environmentally friendly animals:
Phytase transgenic pigs which are capable of expressing bacterial phytase in saliva and thereby reducing fecal phosphorous output have been developed for commercial production in Canada (Golovan et al. 2001).
Biomedical applications: “the mouse is no human”
Even though the laboratory mouse remains a powerhouse genetic model species, the emphasis on using alternative animal model species for biomedical investigation has gained momentum due in part to several findings in which the mouse models and the resulting phenotypes (regarding modeling human biology) have been shown to be confounded and/or overly dependent on the genetic background or strain of mouse used in the study. For example, strain-specific differences (Harris and Juriloff 2010) have been reported to result in different neural tube closure phenotypes in mutants of: Casp3 (Houde et al. 2004), Sod2 (Huang et al. 2006), Egfr (Threadgill et al. 1995), Nnt (Nicholson et al. 2010), Bcl2l2 (Navarro et al. 2012), and associated mutants. In so far as most mouse studies involve inbred mouse strains (to help reduce variability in the response), almost all the domestic animals are crossbreeds, and the resulting phenotype from these animal models is more reliable and applicable to the human disease. An often cited example is that of cystic fibrosis, where mice carrying human mutations do not show the full panoply of symptoms associated with human cystic fibrosis (Carvalho-Oliveira et al. 2007; Grubb and Boucher 1999; Montier et al. 2004; Wilke et al. 2011); the same is true for other diseases such as Lesch-Nylan (Wu and Melton 1993) and Huntington’s Disease (Li and Li 2012; Morton and Howland 2013). As a result, focus is increasingly directed to larger animals with pigs and sheep seeing the greatest use (Casal and Haskins 2006) besides primates (Camus et al. 2015).
In addition to failing to replicate disease mutations, the mouse has both size and physiological limitations and thus inherently cannot fulfill the full spectrum of biomedical needs to address all human disease e.g. preventive medicine (obesity, infertility, cardiovascular disorders), prosthetics, identification of new or improved diagnostics, development of novel therapeutic strategies, and farm-derived zoonotic diseases. These and other examples underscore the need for alternative model species for biomedical investigation. For a comprehensive review of alternative large animal biomedical models, the readers are referred to other recent Review articles (Meurens et al. 2012; Petersen and Niemann 2015; Prather et al. 2008; Swindle et al. 2012; Tan et al. 2016; Whitelaw et al. 2016; Whyte and Prather 2011).
In addition to serving as models of human disease, domesticated animals are coveted for studies where the relatively long-life span, close similarity in body size, and physiology offer an advantage. Briefly, pigs are preferred models for nutritional studies and are a model of choice for investigation of nutrient uptake, trafficking, and metabolism (Patterson et al. 2008). Cattle have played a major role and still are a preferred model for development of in vitro fertilization, embryo culture in vitro, and embryo transfer procedures (Betteridge 2003). Sheep models have pioneered cloning technologies (Campbell et al. 1996) and have been invaluable for modeling human pregnancy and fetal development (Barry and Anthony 2008). They have also served as a model for research on cardiovascular disease and the toxicity of anesthetics (Scheerlinck et al. 2008). Chickens are used as a model for developmental fate mapping (Tickle 2004) and growth physiology (Porter et al. 1995). Taken together, depending on the biological question that needs to be addressed, a suitable or even a preferred large animal model is available for investigation. However, until now, there has been a lack of incentive for the use of large animals as “preferred models”, due to the relatively high costs of maintenance, a lack of suitable funding mechanisms, and genetic modification technologies that lag behind the mouse models. As discussed above, large animal research has entered an exciting phase; the availability of editors for precise alteration of the genome coupled with genome annotation and bio-informatics tools are now affording hitherto unavailable opportunities. In the same way that the genetically modified mouse catapulted mammalian biology to the forefront of biomedical research 30 years ago, we are now positioned to using comparative biology with large animals to further refine the gaps left by the murine era.
Summary: way forward
Animal agriculture is presented with unprecedented challenges. In addition to disease susceptibility and production issues, farmers are now faced with rising animal feed costs, regulatory hurdles including reducing growth promoting antibiotics in animal feed, animal welfare issues, and misguided consumer perceptions (to name a few). With burgeoning global population, ensuring food security and improving production efficiencies are top priorities that need to be dealt with urgently. Intensification of animal agriculture is the likely path forward and associated issues like animal pollution, food safety, and antibiotic usage while maintaining high animal output are topics that will need scientific input now and for years to come. Practices related to animal welfare such as castration and dehorning that are often vilified and are a poster issue for animal welfare organizations are similarly going to play on the public conscience and likely best dealt via novel strain construction rather than relying solely on better animal management tools. Finally, the risks of global pandemics affecting animals, such as foot-and-mouth virus, and zoonotic diseases that affect both the humans and animals alike, e.g. influenza, are other challenges where animal scientists and biomedical scientists will need to join forces. We are at the intersection of genomics and genetics, where causative genes, mutations, and polymorphisms associated with these unwanted traits are easier to identify and can often be eliminated. These modifications can be achieved such that hard earned genetic gains are not lost at the expense of selection for an individual (production or disease resistance) trait. Arguably, most if not all the outlined challenges can be addressed by genetic engineering, specifically precision editing techniques enabled by editors. A better understanding of issues related to large animal health and production are no doubt going to benefit humans in the long term (e.g. health, fertility, obesity). Investment into animal research, in this case, the livestock will have an added advantage of advancing veterinary medicine in addition to human medicine and will prove to be a better investment of resources.
The primary author was supported by Agriculture and Food Research Initiative Competitive Grant # 2015-67015-22845 from the USDA National Institute of Food and Agriculture, and Maryland Agricultural Experiment Station.
Compliance with ethical standards
Conflict of interest
Drs. Bhanu Telugu and Ki-Eun Park are co-founders of RenOVAte Biosciences Inc, a large animal genome editing company.
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