Genome editing for disease resistance in pigs and chickens

• Genome editing technology enlarges the tool box of trait-selective breeding. • Methods for genome editing have developed over the past decades, making the technology more efficient and specific. • Technology to generate edited pigs and chickens is developing alongside genome editors to generate animals faster and more affordable. • For two major pig diseases, it has been shown that resistant animals can be generated that are refractory to infection. In chickens there are promising laboratory results but no genome-edited, resistant chickens yet. • Genome editing allows us to overcome bottlenecks in trait-selective breeding and allows the incorporation of genetic traits from other breeds, related species, or laboratory results. • Two major hurdles still to be faced prior to the implementation of this promising technology are consumer acceptance and the regulatory framework. For thousands of years, humans have used selective breeding to improve desirable traits in both livestock and companion animals. In livestock, targeted breeding has been common practice since the British Agricultural Revolution of the 18th century, with measurable production traits such as feed conversion in cattle or wool production in sheep actively selected for. In the late 20th century, genomic selection was added to the livestock breeding tool box; by reading specific locations in the genome and assigning them to measurable production traits, faster improvement in livestock production efficiency has been achieved. One of the inherently difficult production traits to measure is resistance to a specific disease, as animals with less severe symptoms or pathology may simply have been exposed to less pathogen. Experimental infections guaranteeing equal pathogen exposures are expensive and require large numbers of animals for genetic association studies, making them ethically questionable. Genome editing offers new opportunities to livestock breeding for disease resistance, allowing the direct translation of laboratory research into disease-resistant or resilient animals. Genome editors are custom enzymes that allow scientists to cut the DNA strands in the nucleus of a cell at a specific position. The researcher can then influence how the DNA is repaired, introducing very precise genetic changes at a target locus in their species of interest. This technology has been revolutionary and provides exciting possibilities for the production of livestock resistant to viral diseases. Such opportunities are particularly pertinent given state efforts to improve global food security and reduce food waste throughout the production chain. The most prominent editor technology today, CRISPR/Cas, uses a 20 nucleotide RNA guide to target its enzyme component to a designated locus in the genome. The probability of off-target cutting with a high fidelity Cas enzyme is very low, because with four potential base combinations at each of the 20 nucleotides there are over one trillion unique guide combinations. Once the enzyme has cut the DNA strands, the predominant repair pathway in most cells is nonhomologous end joining, an error-prone process which often introduces small insertions or deletions into the genetic code at the break site. If the target is within a gene, such perturbations can result in a disruption to the function of that gene, potentially leading to a loss of protein function. This can be very useful to basic science as it allows researchers to discover functions associated with novel genes. For many applications, a more precise change to the genome is required. To that end, scientists regularly make an alternative DNA repair process called homology-directed repair. To do this, researchers provide a novel DNA sequence alongside the CRISPR/Cas reagents, whereby the cellular repair machinery uses the new DNA as a template when fixing the break. This approach facilitates the introduction of defined changes at the genomic target locus and has sufficient refinement to alter a single nucleotide, allowing precise modification of gene function. Finally, by introducing a pair of editors, it is possible to generate two concurrent DNA breaks on the same chromosome. The cellular repair machinery then joins the ends of the cut sites, promoting the deletion of the intervening sequence. All the editor reagents introduced to the cell are rapidly degraded, with only the alteration to the genomic sequence remaining to be propagated following cell division. Genome editing has been applied to a wide variety of agricultural species including salmonids, poultry, and ruminants. However, due to its global economic value, relatively short generation time, and multiparous nature, the most edited livestock species to date is the pig. There are two main methods widely used for the generation of edited pigs: cloning of edited fibroblasts or direct injection of the zygotes with editor reagents. Both work well, and each has specific advantages. In cloning, fibroblast cells can be maintained in the lab for prolonged periods. This allows researchers to introduce editor reagents into the cultured cells typically by lipofection, electroporation, or microinjection. Editing events in each cell of a population can be characterized and individual cells with the desired alteration to their genome selected for the cloning process, whereby the fibroblast cell is fused with an enucleated oocyte shell in a process called somatic cell nuclear transfer (Figure 1A). The reconstituted “zygote” is then transferred to a recipient gilt or sow (Carlson et al., 2012). Despite the benefit of being able to prescreen the donor cells, cloning is generally inefficient with hundreds of reconstituted zygotes being transferred to a single recipient. Cloning also yields reduced litter sizes when compared with standard breeding and offspring often demonstrate reduced viability. Methods to generate genome-edited pigs and chickens. (A–C) Editing in pigs. (A) Somatic cell nuclear transfer or more commonly known as cloning. Genome editing is performed in fibroblast cells, which are cultivated in vitro. A specific edit can be selected for before transfer of the nucleus into an enucleated oocyte before transfer to a recipient gilt or sow. (B) Ex vivo ovary (often slaughterhouse-derived) derived oocytes are fertilized in vitro to yield zygotes for editing or zygotes harvested from donor gilts or sows. Genome editing reagents are microinjected into the zygotes and transferred to a recipient. (C) Spermatogonial stem cells are isolated, cultivated, and edited in vitro prior to transfer to a surrogate sire. Heterozygous offspring often need to be mated before yielding a resistant pig. (D–F) Editing in chickens. (D) Electroporation in ovo. Genome editing reagents are electroporated into the embryo in ovo. Resulting chickens are often mosaic and breeding of resistant chickens is only possible if the germ cells are edited. (E) Editing of sperm by lipofection or electroporation can generate heterozygous offspring following artificial insemination. They often have to be mated to yield, homozygous chickens. (F) Primordial germ cells are isolated from embryos, cultivated, and edited in vitro. Selected edited cells are transferred to the blood stream of an embryo where they migrate to the gonad and develop into germ cells. Breeding with the resulting offspring is required to generate homozygous chickens. As an alternative to cloning, newly fertilized zygotes can be directly microinjected with genome-editing reagents and transferred immediately back to the oviduct of a recipient animal (Figure 1B). In contrast to cloning, this approach (Lillico et al., 2013) results in the efficient establishment of pregnancies and robust litters. However, without the prescreening of cells that is routine in cloning, offspring from direct zygote manipulation inevitably encompasses a range of editing outcomes, since selection of a specific edit is not possible. Porcine zygotes can also be generated by maturation of oocytes extracted from slaughterhouse-derived ovaries and in vitro fertilization. Unfortunately, in vitro fertilization in pigs often results in polyspermy, rendering the resulting embryo inviable. However, in this controlled environment, editing rates can be increased and costs and animal use reduced. An emerging alternative to these proven methods could be the use of surrogate sires. As a first step towards this goal, pigs have been edited to remove a gene required for male fertility, generating an empty spermatagonial stem cell niche in the testis (Park et al., 2017). Spermatogonial stem cells can be isolated and cultured in vitro, opening the possibility to edit and characterize these cells before transfer to a recipient (Park et al., 2019) (Figure 1C). Genetic modification of poultry poses unique challenges due to the very different physiology of the avian egg compared with a mammalian oocyte. As a result, isolation and transfer of a chicken yolk is not practical. One approach that has been taken is in ovo electroporation of editing reagents, which allowed the analysis of gene function in the neural crest (Gandhi et al., 2017). However, others reported that electroporation resulted in mosaicism with editing limited to a subset of cells as the chicken embryo is already much further developed when an egg is laid compared with a zygote (Veron et al., 2015) (Figure 1D). As a result, it is unlikely that this approach could be efficiently utilized to generate edited birds. An alternative approach involves sperm transfection–assisted gene editing, whereby sperm are lipofected with editing reagents before use in artificial insemination (Cooper et al., 2017) (Figure 1E). However, advances in chicken stem cell technology show the greatest promise for genome editing in chicken. Primordial germ cells (stem cells that eventually develop into germ cells) can be isolated from the blood of developing chicks in ovo and cultured in vitro. As with mammalian fibroblasts, these cells can be edited and selected in vitro before transfer into the bloodstream of a stage-matched recipient where they migrate to and populate the developing gonad. The chicken embryo is accessed through an opening in the egg shell, which is sealed again until the chicken hatches. Genome editing in primordial germ cells has been successfully demonstrated by a number of groups (Park et al., 2014; Taylor et al., 2017; Idoko-Akoh et al., 2018) and one group has generated modified birds (Park et al., 2014). The founder birds generated from this editing method are chimeric due to the presence of preexisting germ cells. The resulting offspring generated from breeding with the founders will be a mixture of edited or nonedited. Recipient chicken embryos devoid of germ cells are currently being developed that will significantly increase the efficiency of this process (M. McGrew, unpublished results) (Figure 1F). Genome editors will undoubtedly have a significant role on the generation of disease-resistant animals as exemplified below. It is important to note that currently the technology is limited to modifying a single gene or a SNP with large effects; however, disease resistance in many cases is likely to be a polygenic trait. Multiplexing technology is under development such that in the future polygenic traits could be altered in a single step. Porcine reproductive and respiratory syndrome (PRRS) is arguably the most economically important pig disease worldwide. The causative agent of PRRS is an arterivirus, named PRRS virus (PRRSV), that affects pigs of all ages but most importantly causes late-term abortions and stillbirth in sows and severe respiratory disease in piglets with severe morbidity and high mortality. PRRSV also incapacitates the pig’s immune response, providing an ideal breeding ground for severe secondary infections, mostly by bacteria, which leads to increased use of antibiotics. PRRSV exclusively infects cells of the monocyte/macrophage lineage and two macrophage-specific proteins, CD163 and CD169, were identified as receptors for the virus: CD169 acting on the surface of the cells and CD163 inside the internalizing transport vesicles (Calvert et al., 2007; Van Gorp et al., 2008). The virus was thought to attach to CD169 to be taken up into the cells; however, genome-edited pigs lacking CD169 were not resistant to PRRSV infection (Prather et al., 2013). CD163 on the other hand is thought to act through a key-lock interaction with the virus to allow it to escape from the internalizing transport vehicles into the cytosol where it replicates. CD163 consists of nine globular domains, organized like beads on a string, with domain 5 determined to mediate the key-lock interaction allowing viral entry into pig cells (Van Gorp et al., 2010). Using genome editing to generate pigs lacking CD163 Whitworth et al. showed for the first time that this approach could be used to produce livestock resistant to important viral diseases, in this case PRRS (Whitworth et al., 2016). CD163 is known to have a range of important biological functions in homeostasis, inflammation, and immune responses. As a refinement on functional knock out of the entire CD163 protein, editing reagents were designed to remove only domain 5 leaving the remainder of the protein intact. The resulting animals were completely resistant to PRRSV infection and maintained the biological functions associated with the remaining domains of CD163 (Burkard et al., 2017; Burkard et al., 2018). The two coronaviruses porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) both cause severe diarrhea in preweaned piglets and are associated with high morbidity and mortality. In vitro host–pathogen studies identified aminopeptidase N as the receptor for TGEV and a potential receptor for PEDV (Delmas et al., 1992; Li et al., 2007). The use of genome editing to generate pigs lacking aminopeptidase N successfully showed that pigs resistant to TGEV infection could be generated. However, the edited animals remained susceptible to PEDV infection (Whitworth et al., 2019). Aminopeptidase N is important for peptide digestion in the small intestine and knockout mice were shown to have delayed mammary gland development. In humans, aminopeptidase N defects are associated with different types of leukemia and lymphoma. Therefore, further investigation into the potential consequences of the absence aminopeptidase N in pigs is warranted as it may affect the overall health and/or productivity of the animals. African swine fever virus (ASFV) causes a severe hemorrhagic disease in domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa ferus) with high mortality in pigs of all ages. 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