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Title: Production of germline ablated male pigs via Crispr/Cas editing of the NANOS2 gene

item KAUCHER, ANY - Washington State University
item PARK, KI-EUN - University Of Maryland
item Powell, Anne
item OATLEY, MELISSA - Washington State University
item Donovan, David
item Blomberg, Le Ann
item LILLICO, SIMON - University Of Edinburgh
item WHITLAW, BRUCE - University Of Edinburgh
item MILEHAM, ALAN - Genus
item TELUGU, BHANU - University Of Maryland
item OATLEY, JON - University Of Washington

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/12/2017
Publication Date: 3/20/2017
Citation: Kaucher, A., Park, K., Powell, A.M., Oatley, M., Donovan, D.M., Blomberg, L., Lillico, S., Whitlaw, B., Mileham, A., Telugu, B., Oatley, J. 2017. Production of germline ablated male pigs via Crispr/Cas editing of the NANOS2 gene. Meeting Abstract. 10;7:40176. doi: 10.1038/srep40176..

Interpretive Summary:

Technical Abstract: The availability of alternative models to flies, worms, and mice for studying germ cell biology is important for translating findings to higher order mammals. In this context, investigations in pigs and other livestock species can also serve to find applications for both basic biomedical research as well as for agriculture. The goal of this study is to investigate whether the role of NANOS2 is conserved in higher order mammals, and to generate pigs that lack germline but retain an intact soma in males. The germline ablated male pigs are expected to serve as ideal recipients for spermatogonial stem cell transplantation and for expanding the availability of gametes from desirable sires. Targeted inactivation of genes essential for germ cell survival in livestock species is impractical using embryonic stem (ES) cells because of the lack of proven chimera-competent ES cell lines, or arduous and technically demanding requiring somatic cell nuclear transfer or cloning. The recent development of CRISPR/Cas based gene editing allows for creation of double strand breaks at the target site, and introduction of random insertions or deletions, and generation of knockout animals in one generation by microinjection into zygotes. Here, we attempted to generate NANOS2 null male pigs by microinjection of in vitro transcribed, Streptococcus pyogenes (Sp) Cas9 and single guide (sgRNA) targeting NANOS2 into in vivo derived porcine 1- cell embryos. Following microinjection and transplantation into synchronized surrogate pigs, 18 live piglets were obtained with an array of genotypes. All surviving piglets were found to be edited, with eight animals showing biallelic frameshift mutations. All edited animals were viable and matured normally. Examination of cross-sections from biopsies of testicular tissue revealed complete lack of germline in homozygous knockout males but the presence of germ cells and spermatogenesis in heterozygous males. Lack of sperm in the ejaculates from homozygous knockout males confirmed the sterile phenotype. Although the germline was lacking in knockout males, the seminiferous tubules and interstitial tissue appeared intact. On the other hand, NANOS2 null females had intact follicles, and a regular estrous cycle. Collectively, the reproductive parameters in NANOS2 knockout pigs phenocopies Nanos2 null mice demonstrating evolutionary conservation in the role of Nanos2 for development of the spermatogenic lineage. Furthermore, the creation of this NANOS2 mutant line of pigs provides a novel mammalian model to study male germline development and a potential recipient model for expansion of the germline from valuable donor males via spermatogonial stem cell transplantation.