Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9

Area of Science:

• Stem Cell Biology
• Molecular Genetics
• Gene Editing Technologies

Background:

• Precise temporal control of gene expression or deletion is crucial for understanding gene function.
• Establishing human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) is technically challenging.

Purpose of the Study:

• To develop an efficient strategy for generating iKO hPSC lines.
• To enable precise temporal control of gene deletion in hPSCs for functional studies.

Main Methods:

• Combined CRISPR/Cas9 genome editing with Flp/FRT and Cre/LoxP systems.
• Utilized a dual-sgRNA targeting approach for biallelic knockin of FRT sequences.
• Developed a two-step strategy for simultaneous insertion of a recombinase cassette and removal of drug-resistance genes.

Main Results:

• Successfully established iKO hPSC lines (hESC and iPSC) for multiple genes (SOX2, PAX6, OTX2, AGO2).
• Demonstrated the efficiency of the dual-sgRNA targeting and two-step strategy.
• Generated iKO lines for genes with diverse structural and expression patterns.

Conclusions:

• The developed strategy significantly speeds up the generation of iKO hPSC lines.
• Availability of these iKO hPSC lines will transform gene function analysis in human cells.
• This method provides a powerful tool for studying gene function in developmental and disease contexts.