CRISPR/Cas9-Based Safe-Harbor Gene Editing in Rhesus iPSCs

Area of Science:

• Stem cell biology
• Gene editing technologies
• Non-human primate models

Background:

• Induced pluripotent stem cells (iPSCs) are crucial for developing cell-based therapies.
• Monitoring iPSC-derived cells in vivo is essential for assessing safety and efficacy.
• Non-human primate (NHP) models offer clinical relevance for testing these therapies.

Purpose of the Study:

• To develop a safe and efficient method for labeling NHP iPSCs for in vivo tracking.
• To enable targeted gene insertion into genomic safe harbors (GSHs) in RhiPSCs.
• To provide a detailed protocol for genetic modification and clone screening.

Main Methods:

• Utilized CRISPR/Cas9 gene editing to target the Adeno-Associated Virus site 1 (AAVS1) GSH in rhesus macaque induced pluripotent stem cells (RhiPSCs).
• Developed protocols for targeted marker or therapeutic gene insertion.
• Included methods for screening targeted clones and assessing potential off-target nuclease activity.

Main Results:

• Successfully demonstrated targeted gene insertion into the AAVS1 locus in RhiPSCs.
• Provided detailed screening procedures for identifying correctly modified clones.
• Offered a tool for evaluating potential nuclease off-target effects.

Conclusions:

• CRISPR/Cas9 targeting of the AAVS1 GSH is a viable strategy for labeling RhiPSCs.
• This method supports reliable transgene expression for in vivo monitoring.
• The protocol facilitates the advancement of iPSC-based therapies in NHP models.