CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs

Area of Science:

• Stem Cell Biology
• Molecular Genetics
• Biotechnology

Background:

• Human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs), hold immense therapeutic potential.
• Precise, locus-specific, and multiplex genome editing is crucial for unlocking this potential.
• CRISPR/Cas9 technology has emerged as a powerful tool for efficient and versatile genome manipulation.

Purpose of the Study:

• To provide a detailed protocol for generating gene knockout and/or knockin iPSCs.
• To establish a robust method for precise genome editing in hPSCs.
• To facilitate the use of iPSCs in various research and therapeutic applications.

Main Methods:

• Design of guide RNAs (gRNAs) for targeted DNA cleavage.
• CRISPR/Cas9-mediated genome editing using ribonucleoprotein complexes.
• DNA electroporation for introducing editing components into iPSCs.
• T7 endonuclease assay for detecting on-target editing events.
• Single-cell cloning for selecting edited iPSC clones.

Main Results:

• Successful generation of gene knockout and/or knockin iPSCs.
• Demonstration of precise, locus-specific genome editing capabilities.
• Validation of the T7 endonuclease assay for confirming editing events.

Conclusions:

• The developed protocol enables efficient and accurate genome editing in iPSCs.
• This method provides a valuable tool for advancing stem cell research and regenerative medicine.
• Precise genome editing in hPSCs is achievable and essential for future applications.