DNA-guided CRISPR/Cas12 for RNA targeting.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas nucleases are powerful tools for genome editing, RNA editing, and diagnostics.
• Current CRISPR systems primarily rely on RNA-guided mechanisms, limiting their versatility.

Purpose of the Study:

• To introduce ΨDNA, a novel DNA-based guide for Cas12 enzymes, for efficient and specific RNA targeting.
• To explore the potential of ΨDNA in diagnostic applications and cellular RNA modulation.

Main Methods:

• Engineered ΨDNA molecules that mimic crRNA in a reverse orientation for Cas12-RNA assembly.
• Tested ΨDNA for RNA sensing capabilities, including short and long RNA detection.
• Evaluated ΨDNA's performance in detecting Hepatitis C Virus (HCV) RNA in clinical samples.
• Investigated ΨDNA's ability to guide Cas12 enzymes for RNA targeting within cells, including mRNA degradation and multiplex knockdown.

Main Results:

• ΨDNA demonstrated stable Cas12-RNA assembly and activated trans-cleavage activity without RNA components.
• ΨDNAs showed high efficacy in sensing both short and long RNA targets.
• Achieved 100% accuracy in detecting HCV RNA from clinical samples.
• Successfully guided Cas12 enzymes for RNA targeting in cellular environments, leading to enhanced mRNA degradation and multiplex RNA transcript knockdown.

Conclusions:

• ΨDNA represents a robust DNA-based alternative to traditional RNA guides for CRISPR-Cas12 systems.
• This technology significantly expands the potential of CRISPR-Cas12 for advanced diagnostic tools.
• ΨDNA enables targeted RNA modulation within cellular contexts, offering new therapeutic and research avenues.