Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells.

Area of Science:

• Biotechnology
• Stem Cell Biology
• Genetics

Background:

• Human pluripotent stem cells (hPSCs) are crucial for disease modeling, drug screening, and regenerative medicine.
• CRISPR/Cas9 technology offers powerful genome editing capabilities for hPSCs.
• Accessibility to robust hPSC genome editing protocols is limited for many research groups.

Purpose of the Study:

• To create a comprehensive resource for performing CRISPR/Cas9 genome editing in hPSCs.
• To empower researchers with varying experience levels in hPSC culture and gene editing.
• To facilitate the generation of precisely modified hPSC lines for diverse applications.

Main Methods:

• Detailed protocols for generating gene knock-outs in hPSCs.
• Methods for introducing small, targeted mutations into the hPSC genome.
• Protocols for creating hPSC lines with knock-in reporter genes.

Main Results:

• Successful generation of hPSC lines with specific gene knock-outs.
• Demonstrated ability to introduce targeted mutations accurately.
• Established methods for knock-in reporter gene integration into hPSCs.

Conclusions:

• The developed resource simplifies CRISPR/Cas9 genome editing in hPSCs.
• This work supports advancements in hPSC-based disease modeling and therapeutic strategies.
• The provided theoretical framework allows for adaptation to future gene-editing innovations.