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.Tal-effector nuclease | biotechnology | gene-editing

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

In Trypanosoma brucei, DNA recombination is crucial in antigenic variation, a strategy for evading the mammalian host immune system found in a wide variety of pathogens. T.brucei has the capacity to encode >1000 antigenically distinct variant surface glycoproteins (VSGs). By ensuring that only one VSG is expressed on the cell surface at one time, and by periodically switching the VSG gene that is expressed, T.brucei can evade immune killing for prolonged periods. Much of VSG switching appears to rely on a widely conserved DNA repair pathway called homologous recombination, driven by RAD51. Here, we demonstrate that T.brucei encodes a further five RAD51-related proteins, more than has been identified in other single-celled eukaryotes to date. We have investigated the roles of two of the RAD51-related proteins in T.brucei, and show that they contribute to DNA repair, homologous recombination and RAD51 function in the cell. Surprisingly, however, only one of the two proteins contributes to VSG switching, suggesting that the family of diverged RAD51 proteins present in T.brucei have assumed specialized functions in homologous recombination, analogous to related proteins in metazoan eukaryotes.