KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Drug Discovery

Background:

• CRISPR-Cas9 genome engineering tools can cause unintended off-target mutations.
• Existing methods to control Cas9 activity involve protein or small-molecule inhibitors.
• Modulating Cas9 activity is crucial for improving the safety and precision of gene editing.

Purpose of the Study:

• To identify novel small-molecule modulators of CRISPR-Cas9 activity.
• To investigate the mechanism of action of identified modulators.
• To assess the potential of these modulators in enhancing CRISPR-Cas9 specificity.

Main Methods:

• Screening of small molecule compounds with irreversible warheads.
• Assessing cellular activity of Cas9 in genome-, base-, and prime-editing tools.
• Investigating the interaction of modulators with Cas9 and its mRNA.
• Evaluating the effect of modulators on CRISPR-Cas9 specificity in human cells.

Main Results:

• Selective inhibitors of nuclear export (SINEs) were identified as potent inhibitors of cellular Cas9 activity.
• SINEs function indirectly by interfering with Cas9 mRNA nuclear export, not by direct Cas9 inhibition.
• SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9.
• An FDA-approved drug, KPT330, and other SINEs significantly improved the specificity of CRISPR-Cas9 genome and base editing in human cells.

Conclusions:

• SINEs offer a novel strategy for modulating CRISPR-Cas9 activity.
• These compounds provide an effective means to enhance the specificity of CRISPR-Cas9-based genome engineering.
• The findings expand the available tools for controlling CRISPR-Cas9 and improving its therapeutic potential.