Dithiothreitol Facilitates LbCas12a with Expanded PAM Preference for Ultrasensitive Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Nucleic Acid Detection

Background:

• CRISPR/Cas systems offer high specificity for nucleic acid targeting in molecular diagnostics.
• A key limitation is the requirement for a protospacer adjacent motif (PAM) for Cas protein activity, restricting random detection.
• The LbCas12a protein's reliance on a specific PAM sequence hinders its application in diverse diagnostic scenarios.

Purpose of the Study:

• To overcome the PAM specificity limitation of CRISPR/Cas12a for enhanced nucleic acid detection.
• To develop a DTT-mediated CRISPR/Cas12a system with relaxed PAM specificity.
• To demonstrate the utility of this new system for sensitive and selective detection of high-risk human papillomavirus (HPV).

Main Methods:

• Utilized dithiothreitol (DTT) to induce relaxed PAM specificity in LbCas12a.
• Integrated the DTT-modified CRISPR/Cas12a system with nucleic acid amplification techniques (PCR, RPA).
• Developed a diagnostic platform for detecting HPV 16 and 18, incorporating lateral flow assays (LFA) for point-of-care applications.

Main Results:

• The DTT-mediated CRISPR/Cas12a system (DTT-deCRISPR) demonstrated relaxed PAM specificity.
• Achieved sensitive detection of synthesized HPV 16 and 18 plasmids down to 1 aM within 60 minutes.
• Clinical sensitivity for HPV 16 and 18 detection reached 93.75% and 80.00%, respectively, with LFA results correlating well with fluorescence analysis.

Conclusions:

• Dithiothreitol (DTT) unexpectedly modulates LbCas12a's PAM preference, expanding its utility.
• The DTT-deCRISPR system offers enhanced universality and flexibility for CRISPR-based molecular diagnostics.
• This technology shows promise for sensitive, rapid, and point-of-care detection of pathogens like HPV.