Resources for genome editing in livestock: Cas9-expressing chickens and pigs

Abstract

43Genetically modified animals continue to provide important insights in biomedical sciences. 44 Research has focused mostly on genetically modified mice so far, but other species like pigs 45 resemble more closely the human physiology. In addition, cross-species comparisons with 46 phylogenetically distant species such as chickens provide powerful insights into fundamental 47 biological and biomedical processes. One of the most versatile genetic methods applicable across 48 species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic 49 chickens and pigs that allow in vivo genome editing in these two important agricultural species. 50We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the 51 animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number 52 of different target genes and for a variety of cell types. Taken together, these transgenic animal 53 species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, 54 and will facilitate organ specific reverse genetics as well as cross-species comparisons. 55Significance statement 56 Genome engineering of animals is crucial for translational medicine and the study of genetic traits. 57Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, 58 providing the basis for in vivo genome editing. We demonstrated the functionality of this system 59 3 by successful genome editing in chicken and porcine cells and tissues. These animals facilitate 60 organ specific in vivo genome editing in both species without laborious germ line modifications, 61 which will reduce the number of animals needed for genetic studies. They also provide a new tool 62 for functional genomics, developmental biology and numerous other applications in biomedical 63 and agricultural science.64 Introduction 65 Chickens and pigs are the most important livestock species worldwide. They are not only important 66 sources of food, but also valuable models for evolutionary biology and biomedical science. Pigs 67 share a high anatomical and physiological similarity with humans, and are an important species for 68 translational biomedical research e.g. in the areas of cancer, diabetes, neurodegenerative and 69 cardiovascular diseases (1-3). In contrast, chickens are phylogenetically distant vertebrates from 70 humans, but they were instrumental in the field of developmental biology due to the easy access to 71 the embryonated egg. They are used to study neurological and cardiovascular functions (4-6) and 72 provided key findings in B cell development and graft versus host responses (7-9). 73 Modelling human diseases in animals helps elucidating disease pathways and enables the 74 development of new therapies. Although mice are an intensively studied vertebrate model (10), 75 they are often not optimal for modelling particular human diseases. For example, mouse models 76 for familiar adenomatous polyposis (FAP) poo...

Genome editing of avian species: implications for animal use and welfare

“…[28][29][30] In an improvement of this technique, Bartsch et al combined Cas9 transgenic chicken embryos with the in ovo electroporation of guide RNAs, demonstrating in ovo electroporation in the chicken embryo is a feasible method of generating inovo GE chickens. 31 Similar experiments carried out in mammalian species require surgery for the in utero manipulations of the embryo with concomitant increased welfare impacts on the mother. Thus, the use of avian embryos represents a refinement by reducing the number of animals used and carrying out experiments at unprotected stages.…”