Beate Rieblinger, Hicham Sid, Denise Duda, Tarik Bozoglu, Romina Klinger, Antonina Schlickenrieder, Kamila Lengyel, Krzysztof Flisikowski, Tatiana Flisikowska, Nina Simm, Alessandro Grodziecki, Carolin Perleberg, Andrea Bähr, Lucie Carrier, Mayuko Kurome, Valeri Zakhartchenko, Barbara Kessler, Eckhard Wolf, Lutz Kettler, Harald Luksch, Ibrahim T. Hagag, Daniel Wise, Jim Kaufman, Benedikt B. Kaufer, Christian Kupatt, Angelika Schnieke, and Benjamin Schusser

PNAS March 9, 2021 118 (10) e2022562118

Significance

Genetically modified animals are crucial for functional studies and translational biomedical research. However, the generation of genome-edited large animals is time consuming and inefficient. Here, we report the generation of transgenic pigs and chickens that ubiquitously express Cas9 nuclease. The functionality of Cas9 was demonstrated for different target genes, a variety of cell types, and in vivo for the heart and the developing brain. Genome editing can now easily be carried out in vivo in specific organs or tissues without the need to generate germline-modified animals. These Cas9 transgenic animals provide an innovative and efficient resource for in vivo genome editing in biomedical and agricultural sciences.

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.

See: https://www.pnas.org/content/118/10/e2022562118

Figure 1:

Generation and expression analyses of SpCas9 transgenic pigs and chickens. (A) Structure of the ROSA26-SpCas9 targeting vector and targeting strategy to introduce SpCas9 gene into the porcine ROSA26 locus. Exons are indicated by numbered boxes, and regions of homology are indicated by dotted lines. (B) Expression vector used for the generation of SpCas9-expressing PGCs. (C) SpCas9 transgenic founder pigs. (D) SpCas9 transgenic rooster and hen. (E) RT-PCR analyses of SpCas9 transgenic pig organs. SpCas9 expression is shown by a 415-bp PCR product, and porcine GAPDH (576 bp) serves as a control. (F) RT-PCR analyses of SpCas9 transgenic chicken organs. SpCas9 expression in all tissues analyzed is shown by a 265-bp PCR product, and β-actin (300 bp) serves as a control.