A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.

Automated summary

NHPs are valuable animal models for studying clinically relevant mutations due to their close relationship to humans, but the toolbox for genetic modification of NHPs is less developed compared to other species. PSCs share key molecular signatures with the early embryo and are important for validating genome editing approaches. The piggyBac transposon system has been utilized for various purposes in NHP stem cell technology and genome editing, showing advancements in reprogramming, culture, CRISPR/Cas-based genome editing, and establishment of gene-edited monoclonal NHP-iPSC lines.