Genetically engineered (GE) livestock have existed since the mid-1980s, and, since then, a range of methods for delivery of the transgene have been developed, each with advantages and limitations. In regards to the wealth of possible methods for the production of GE animals, two general approaches have emerged. Historically first, the direct manipulation of the zygote (including manipulation of germ cells) now comes in many flavours, from direct pronuclear injection of double-stranded DNA constructs to the use of vectors to deliver the transgene. The second approach utilises an in vitro stage where cells are engineered in culture before being introduced in some way to the developing embryo. The former suffers from lack of control of transgene integration, which can expose the transgene to position effects. The latter can be exploited through homologous recombination to engineer specific genetic loci; with somatic cell cloning being the most widely used method. Both approaches can now be combined with the exciting new editor technologies to enable precise genome editing which in some cases does not involve the incorporation of a transgene. For methods involving the zygote, the use of specific vectors can be of advantage, and the same can be true for the manipulation of cells; however, many delivery strategies are possible for this process. Overall, the drivers for delivery method development have revolved around efficiency and specificity. With regard to viral vectors, and possibly nonviral nanoparticle formulations in the future, spectacular increases in transgenesis rates can be achieved. With the most widely used vector, based on a lentivirus genome, in some cases all animals born from injected zygotes can be transgenic. In livestock, where gestation and breeding times are long, this dramatically reduces the time to proof-of-concept for a given project. In addition, these founder animals will carry different transgene copy-numbers, which is associated with different levels of transgene expression. This strategy can be exploited to quickly produce a large cohort of animals that enable modelling of the range of phenotype observed in a population for a given disease. In addition to the delivery of a transgene, such vectors can also be beneficial for the delivery of reagents that facilitate genome engineering, the most exciting of which are the genome editors. In this situation, either the editor and/or any DNA sequence to be incorporated can be efficiently delivered if not essential for broad uptake of this approach. It is likely that nonintegrating vectors will be desirable. In summary, viral vectors have a broad utility in facilitating the production of GE animals. In the future, nonviral nanoparticles may offer similar opportunities. Given the breadth of methodologies available and with the anticipated use of GE livestock in both agricultural and biomedical applications gaining momentum, we are entering an era of unparalleled opportunity in this area of animal biotechnology.
Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
