Studies on Genome Editing in Avian Species for Acquiring Resistances Against to Avian Leukosis Virus and Site-Specific Recombination

Abstract

Avian species has known as the most suitable model animals in the research fields due to their unique embryonic development. In particular, their reproductive characteristics, such as a relatively short reproductive cycle and laying over hundreds eggs annually, enhance their suitability for this research area. With rapid development of biotechnology, the demand for genome-edited avian species has been also increased. Especially, the recently reported programmed genome editing technology that can induce gene modification at target locus in an efficient and precise manner facilitates establishment of animal models. In this regards, we evaluated these up-to- date biotechnologies including piggyBac transposition, Flipase/Flipase recognition target (Flp/FRT) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) in avian species. Furthermore, we investigated feasibility of semen cryopreservation in chicken for efficient preservation of avian genetic resources including genome-edited avian species. The first study was performed to evaluate the feasibility of CRIPSR/Cas9 system in specific chicken gene, which relating to avian viral disease. Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection. Using same strategy, we also targeted chicken Na+/H+ exchange 1 (chNHE1) gene, which is known as host receptor specifically binds to ALV subgroup J. After first appearance of ALV subgroup J in 1991, the virus causes enormous economic losses in poultry industry. There is no chicken that have resistant against to the virus in nature, the demands for develop resistant chicken lines has been emphasized. The host receptor and the critical amino acid have been identified in avian cells, however, there is no report about acquiring resistant against to the virus mediated by host genome modification. Therefore, in this study, we modified the chNHE1 gene using CRISPR/Cas9 system, and validated the susceptibility of the modified cell lines. As results, we could find that artificially generating premature stop codon on chNHE1 receptor causes absolute resistant to ALV subgroup J, and indel mutations containing W38 is critical. Targeted recombination on W38 regions revealed that deletions only containing W38 are most effective to acquire resistant against to the virus. These results suggest that CRISPR/Cas9-mediated genome replacement could be utilized to precise genome editing in chicken cells and be a efficient tool for developing avian viral disease resistant lines. For the next study, we evaluated recombinase-mediated gene cassette exchange (RMCE) in avian species. Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce RMCE, one of the site-specific recombination technologies, and confirmed RMCE in TG chicken–derived cells. As a result, we established TG chicken lines that have, FRT pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. Next, we investigated the feasibility of semen cryopreservation in chicken to efficiently preserve the avian genetic resources including genome-edited chicken. The importance of genetic resource preservation has been highlighted in the literature as means of maintaining genetic diversity. Among the various methods of preserving such resources, semen cryopreservation can be advantageous because it reduces the time of restoring genetic resources and is less technique-dependent. The Korean Ogye (KO) chicken is a Natural Monument and is recognized as an important genetic resource in Korea. However, successful cryopreservation methods for KO chickens have yet to be reported. Therefore, we completed cryopreservation methods in KO chickens using N-methylacetamide (MA) as a cryoprotectant. Also we performed additional experiments to identify whether fertility and hatchability are affected by long-term storage. Finally, we examined sperm viability in the cryopreserved semen. Our results suggest that the cryopreservation method using MA can be applied to KO chickens regardless of storage period and could be a useful tool for the preservation the endangered avian species. Based on the researches, we identified that the efficient genome editing system mediated by piggyBac transposition, Flp/FRT and CRISPR/Cas9 could be utilized in chicken system. The results suggest that these genome editing technologies can contribute to facilitate the studies on avian species and give us chances to establish novel model for research area as well as industry area. Furthermore, preservation system mediated by semen cryopreservation of this study also will contribute to establish novel chicken lines including genome-edited chicken.