Cas9-expressing chickens and pigs as resources for genome editing in livestock

Abstract

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes

“…livestock. The advent of CRISPR-Cas9 makes transgenesis in different species relatively straightforward, and safe-harbour loci for transgene integration have been described in pigs, cattle and chickens 42 – 44 . Furthermore, transgenesis could be achieved by zygote injection rather than by ES cell targeting.…”

Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid

“…Very promising and versatile resource is the Cas9 stably expressed in all tissues of transgenic animals, which facilitates in vivo organ-specific gene editing. This strategy was recently established in parallel in pigs and chickens [41]. The last addition to the PGC technology in the chicken is the system of inducible germ line ablation and introduction of donor genome-edited PGC into the surrogate host chicken [42].…”

Genome Editing Strategies to Protect Livestock from Viral Infections

“…Recently, we generated pigs carrying Cas9 and a gRNA-targeting ASFV and observed no abnormalities in the animals (unpublished). Cas9-expressing pigs were healthy and showed no reproductive impairments [123,124]. In addition, no toxicity was observed in transgenic chickens expressing Cas9 and gRNAs against MDV [23].…”