Joonbum Lee, Dong-Hwan Kim, Madeline C. Karolak, +1, and Kichoon Lee

https://orcid.org/0000-0001-6169-7516 lee.2626@osu.edu

Edited by Xiang-Jin Meng, Virginia Polytechnic Institute and State University, Blacksburg, VA; received August 21, 2022; accepted October 3, 2022

PNAS November 2, 2022; 119 (45) e2214344119

https://doi.org/10.1073/pnas.2214344119

Abstract

Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.

See https://www.pnas.org/doi/10.1073/pnas.2214344119

Figure 1: Generation of genome-edited chicken and duck lines using the adenovirus-mediated method. The adenovirus containing the CRISPR-Cas9 system is injected into the avian blastoderm. The injected adenovirus in the subgerminal cavity transduces blastodermal cells, including PGCs. Potential germline mosaic founder birds (G0) are hatched from the adenovirus-injected eggs and are utilized for mating with WT birds. Offspring from the founder lines are genotyped, and heterozygous mutant (+/−) males and females (G1) are mated to produce homozygous mutant (−/−) birds (G2).