Dataset Information

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.

SUBMITTER: Rieblinger B

PROVIDER: S-EPMC7958376 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Rieblinger Beate B Sid Hicham H Duda Denise D Bozoglu Tarik T Klinger Romina R Schlickenrieder Antonina A Lengyel Kamila K Flisikowski Krzysztof K Flisikowska Tatiana T Simm Nina N Grodziecki Alessandro A Perleberg Carolin C Bähr Andrea A Carrier Lucie L Kurome Mayuko M Zakhartchenko Valeri V Kessler Barbara B Wolf Eckhard E Kettler Lutz L Luksch Harald H Hagag Ibrahim T IT Wise Daniel D Kaufman Jim J Kaufer Benedikt B BB Kupatt Christian C Schnieke Angelika A Schusser Benjamin B

Proceedings of the National Academy of Sciences of the United States of America 20210301 10

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 CRIS ...[more]

PMID: 33658378

Similar Datasets

Project description:The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets