Development of aptamer-based inhibitors for CRISPR/Cas system.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas9 genome editing technology is powerful but faces challenges with unintended mutations and deletions.
• Controlling excessive or prolonged Cas9 enzyme activity is a key strategy to improve genome editing safety.

Purpose of the Study:

• To develop an inhibitory DNA aptamer targeting the Cas9 enzyme.
• To assess the aptamer's efficacy in controlling Cas9 activity and reducing genome editing errors in vitro and in cells.

Main Methods:

• In vitro selection (SELEX) was used to identify DNA aptamers that bind to Cas9.
• In vitro cleavage assays were performed to evaluate the inhibitory activity of the selected aptamers.
• Modified aptamers incorporating locked nucleic acids were synthesized and tested in cellular assays.

Main Results:

• A DNA aptamer with low nanomolar binding affinity for Cas9 was developed.
• The aptamer demonstrated partial duplex formation with CRISPR RNA, contributing to Cas9 inhibition.
• Modified aptamers significantly suppressed Cas9-directed genome editing and reduced off-target mutations in cellular models.

Conclusions:

• The developed DNA aptamer effectively inhibits Cas9 activity.
• Improving the aptamer with locked nucleic acids enhances its ability to control genome editing and minimize errors.
• This work provides a foundation for developing safer and more controllable CRISPR/Cas9-based gene editing tools for research and therapeutics.