Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Editing

Background:

• CRISPR-Cas9 is a versatile genome-editing technology with broad applications in biomedicine.
• Controlling CRISPR-Cas9 activity in specific cell types remains a challenge for targeted gene editing.

Purpose of the Study:

• To engineer a synthetic RNA-based CRISPR-Cas9 system responsive to endogenous microRNA (miRNA) signatures.
• To achieve cell-type selective genome editing by modulating Cas9 activity based on intracellular miRNA profiles.

Main Methods:

• Developed a microRNA (miRNA)-responsive CRISPR-Cas9 (miR-Cas9) switch by incorporating a miRNA-complementary sequence into the 5'-UTR of Streptococcus pyogenes Cas9 mRNA.
• Tested miR-Cas9 switches for responsiveness to specific miRNAs (miR-21-5p and miR-302a-5p) in distinct cell types (HeLa and human induced pluripotent stem cells).

Main Results:

• The miR-Cas9 switches selectively and efficiently responded to target endogenous miRNAs in specific cell lines.
• Cas9 activity was post-transcriptionally attenuated only in cells expressing the target miRNA, demonstrating cell-type specificity.
• The system allowed for differential control of genome editing by sensing endogenous miRNA activity in heterogeneous cell populations.

Conclusions:

• The developed miR-Cas9 switch system provides a novel framework for cell-type selective genome editing.
• This approach enables sophisticated cell engineering strategies based on endogenous miRNA information for targeted biotechnology and biomedical applications.