An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells

Area of Science:

• Stem cell biology
• Gene editing technologies
• Molecular genetics

Background:

• Human pluripotent stem cells (hPSCs) are crucial for studying complex traits and diseases.
• CRISPR/Cas9 gene editing is a powerful tool for loss-of-function studies in hPSCs.

Purpose of the Study:

• To develop a highly efficient CRISPR/Cas9 system for gene knockout in hPSCs.
• To create a versatile platform for advanced genetic analysis in hPSCs.

Main Methods:

• Development of an episomal vector-based CRISPR/Cas9 system (epiCRISPR).
• Utilized high-fidelity Cas9 and a double-nicking strategy to minimize off-target effects.
• Achieved high Insertion/Deletion (indel) rates.

Main Results:

• The epiCRISPR system demonstrated up to 100% indel rates in hPSCs.
• Successfully enabled efficient double-gene knockout and genomic deletion.
• Minimized off-target cleavage through optimized strategies.

Conclusions:

• The epiCRISPR system provides a highly efficient and precise platform for genetic manipulation in hPSCs.
• Facilitates in-depth genetic analysis for understanding complex traits and diseases using hPSCs.