DNA-guided CRISPR/Cas12 for RNA targeting

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas systems are powerful tools for genome editing, RNA editing, and diagnostics.
• Current CRISPR applications primarily rely on RNA-guided systems, limiting their scope.
• Developing DNA-based guides offers a novel approach to expand CRISPR functionalities.

Purpose of the Study:

• To introduce ΨDNA, a DNA-based guide for Cas12 enzymes, for efficient and specific RNA targeting.
• To evaluate ΨDNA's efficacy in RNA detection and cellular RNA modulation.
• To establish ΨDNA as a viable alternative to RNA guides in CRISPR-Cas12 applications.

Main Methods:

• Engineering ΨDNA, a DNA-based guide mimicking crRNA in reverse orientation, for Cas12 enzymes.
• Assessing Cas12-RNA assembly and trans-cleavage activity without RNA components.
• Testing ΨDNA for short and long RNA sensing, including Hepatitis C Virus (HCV) RNA detection in clinical samples.
• Evaluating ΨDNA's ability to guide Cas12 enzymes for RNA targeting in cellular environments, including mRNA degradation and multiplex knockdown.

Main Results:

• ΨDNA demonstrated stable Cas12-RNA assembly and activated trans-cleavage activity.
• ΨDNAs effectively sensed both short and long RNAs, achieving 100% accuracy in detecting HCV RNA in clinical samples.
• ΨDNAs successfully guided Cas12 enzymes for RNA targeting in cells, leading to enhanced mRNA degradation via ribosome stalling.
• Multiplex knockdown of multiple RNA transcripts was achieved using ΨDNA-guided Cas12 enzymes.

Conclusions:

• ΨDNA serves as a robust DNA-based alternative to traditional RNA guides for CRISPR-Cas12 systems.
• This DNA-guided approach significantly enhances the potential of CRISPR-Cas12 for diagnostic applications.
• ΨDNA enables targeted RNA modulation within cellular environments, opening new avenues for research and therapeutics.