Targeted Gene Editing in Porcine Spermatogonia.

Dennis Webster,Daniel F Carlson,Ina Dobrinski,Staci Solin,Taylor Goldsmith,Lin Su,Alla Bondareva,Nathalia De Lima E Martins Lara

doi:10.3389/fgene.2020.627673

Dennis Webster, Daniel F Carlson + Show 6 more

Open Access

https://doi.org/10.3389/fgene.2020.627673

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Abstract

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

Highlights

Applicable preclinical models are needed to investigate the pathophysiology of human diseases, develop novel treatments and medical devices, and improve approaches for regenerative medicine
As the initial live offspring produced by GST would be heterozygote due to modification of the male germline only, we chose to evaluate gene editing at three biomedically relevant loci associated with either dominant forms of diabetes mellitus, Hepatocyte nuclear factor-1 alpha (HNF1a) and insulin (INS), or a common safe-harbor locus for transgene insertion, ROSA26 (Figure 2A)
Each of the templates was delivered into spermatogonia by CRISPR/Cas9 RNPs using the conditions used for nonhomologous end joining (NHEJ)

Summary

Introduction

Applicable preclinical models are needed to investigate the pathophysiology of human diseases, develop novel treatments and medical devices, and improve approaches for regenerative medicine. While rodent models are currently the standard for early preclinical studies, pigs are physiologically, anatomically, and genetically more similar to humans than are mice and are delivering increasing value to biomedical research. Modified pigs, such as pig models of cystic fibrosis (Rogers et al, 2009), neurofibromatosis type I (Isakson et al, 2018), and diabetes (Kleinwort et al, 2017), are essential where rodent models fail to recapitulate the full pathophysiological spectrum of a disease. There are no molecular markers that allow prospective identification of SSCs within the population of undifferentiated type A spermatogonia

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