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One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these ‘molecular scissors’ have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way—but the most exciting period is just starting.

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.

Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA-binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. TALEN-induced double-strand breaks can be used for gene inactivation via repair by nonhomologous end joining (NHEJ) or to stimulate homologous recombination with a user-specified template to create custom-designed disease alleles. Transfection of multiple TALEN pairs targeting the same chromosome can also produce chromosomal rearrangements such as large inversions and deletions. As genetic modification of primary pig fibroblast followed by cloning is the primary method used to generate transgenic swine, we tested our capacity to mimic multiple common mutation types in primary fibroblasts using TALEN. Either plasmids or mRNA encoding TALEN was transfected into 500 000 to 750 000 fibroblasts by nucleofection followed by 3 days of culture at 30°C before analysis of activity. With this approach, we found that over 65% of custom-designed TALEN displayed activity in pig fibroblasts with typical percentage of indel positive chromosomes ranging from 20 to 45%. Individual clones were derived by either co-selection with an unlinked selection transposon or by dilution cloning. Colonies with mono- and biallelic modification to the target loci were readily identified (up to 84 and 24%, respectively) and could be accomplished without the aid of selection. Co-transfection of 2 TALEN pairs directed to the same chromosome resulted in deletion or inversion of the intervening sequence in 10 and 4% of isolated colonies, respectively. In addition, co-transfection of TALEN with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, 3% of which were homozygous (n = 184 colonies). To prove that modified cells were suitable for generation of animals, we pooled several colonies of Ossabaw fibroblasts harboring TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for cloning. Pregnancy was established in 7/9 transfers (100–150 embryos per transfer), and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Three monoallelic frame-shift genotypes were observed among 9 piglets; as expected, each genotype was represented in the parental cell pool. The remaining 9 animals displayed an identical biallelic mutant genotype and are being investigated as a knockout model of familial hypercholesterolemia (FH). Finally, fibroblasts homozygous for a specified inactivating mutation were created by allele introgression and were cloned, demonstrating that gene editing can be used to create precise swine knock-ins in a single generation.