A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification

Area of Science:

• Molecular Biology
• Biochemistry
• Diagnostics

Background:

• CRISPR/Cas systems offer powerful molecular diagnostics but have limited sensitivity for in situ imaging due to linear amplification.
• Phosphorothioate (PS)-modified nucleic acids can influence enzyme activity through hydrophobic interactions.

Purpose of the Study:

• To engineer a novel CRISPR/Cas amplification strategy for enhanced sensitivity and in situ imaging.
• To investigate the mechanism of PS modification in modulating Cas enzyme activity.
• To develop a new platform for precise molecular diagnostics.

Main Methods:

• Designed linear "Coordinator" probes with "scattered" PS modifications.
• Developed a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA).
• Tested SACA for Cas12a and Cas13a enzyme sensitivity enhancement and in situ imaging of HPV mRNA in cells.

Main Results:

• Achieved exponential amplification without external enzymes, increasing Cas12a sensitivity by 50,000-fold and Cas13a by 10,000-fold.
• Demonstrated superior biostability and structural simplicity of the linear probes.
• Successfully performed precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells.

Conclusions:

• PS modification provides precise control over Cas enzyme activation and trans-cleavage resistance.
• SACA represents a new paradigm for highly sensitive and efficient molecular diagnostics.
• This approach advances the understanding of chemically modified nucleic acid regulation of Cas enzymes.