Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Synthetic Biology

Background:

• CRISPR/Cas9 is a key tool for gene function studies.
• Temporal control of gene editing is crucial for complex biological investigations.
• Existing methods may lack precision or flexibility in temporal control.

Purpose of the Study:

• To develop a method for temporal control of CRISPR/Cas9 activity.
• To enable conditional Cas9 expression and gene editing.
• To facilitate independent manipulation of alleles with Cas9 and other genetic tools.

Main Methods:

• Fusion of an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9).
• Conditional Cas9 expression induced by an FKBP12 synthetic ligand.
• Co-expression of DD-Cas9 with other genes from the same promoter.
• Integration with inducible Cre-ER^T2 for parallel allele manipulation.

Main Results:

• Demonstrated temporal control of CRISPR/Cas9 gene editing.
• Achieved conditional Cas9 expression using DD-Cas9 and a synthetic ligand.
• Showcased adaptability for co-expression without modulating other genes.
• Enabled single-cell specific, parallel manipulation of alleles with Cas9 and Cre-ER^T2.

Conclusions:

• The DD-Cas9 system provides precise temporal control over gene editing.
• This platform offers flexibility for studying gene function and interactions.
• It is valuable for systematic gene characterization and investigating gene networks.