Engineered CRISPR/Cas12a2 Nanoprobe Imaging in Living Cells for Precise Tumor Diagnosis

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Messenger RNA (mRNA) imaging is vital for tumor monitoring but faces challenges in specificity and sensitivity due to intracellular complexity and background noise.
• Current methods struggle to achieve precise mRNA detection within the tumor microenvironment.

Purpose of the Study:

• To engineer a highly specific and sensitive CRISPR/Cas12a2-based system for intracellular mRNA imaging.
• To develop a targeted nanoprobe for distinguishing pancreatic tumor cells from normal cells via mRNA detection.

Main Methods:

• Engineered a CRISPR/Cas12a2 system with a glutathione-responsive RNA blocking strand to control CRISPR/Cas12a2 ribonucleoprotein (RNP) activity.
• Developed a nanoprobe (eRNP-FHR) by integrating the engineered RNP with framework-hotspot reporters (FHR) featuring aptamers targeting tumor cell surface receptors.
• Utilized aptamer-mediated endocytosis for tumor cell targeting and triggered fluorescence release upon specific mRNA recognition and FHR cleavage.

Main Results:

• The eRNP-FHR nanoprobe successfully achieved specific imaging of baculoviral IAP repeat-containing 5 mRNA in pancreatic tumor cells.
• Demonstrated accurate differentiation between pancreatic tumor cells and normal cells using the developed imaging system.
• In a murine pancreatic tumor model, eRNP-FHR showed excellent performance in mRNA imaging, indicating potential for precise tumor diagnosis.

Conclusions:

• The engineered CRISPR/Cas12a2 system and eRNP-FHR nanoprobe offer a promising platform for sensitive and specific mRNA imaging in cancer research.
• This approach holds significant potential for advancing precise tumor diagnosis and monitoring through intracellular mRNA detection.