PAM-free diagnostics with diverse type V CRISPR-Cas systems.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Type V CRISPR-Cas effectors are powerful tools for nucleic acid biomarker detection.
• A major limitation of these systems is their reliance on protospacer adjacent motif (PAM) sequences on target DNA.
• This PAM dependence restricts the application of CRISPR diagnostics.

Approach:

• A novel method, PICNIC (Protospacer-adjacent motif-Independent Nucleic acid detection via CRISPR), was developed.
• PICNIC involves separating double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) using high-temperature and high-pH treatment.
• The released ssDNA is then detected by Cas12 enzymes in a PAM-free manner.

Key Points:

• PICNIC enables PAM-free detection using Cas12a, Cas12b, and Cas12i enzymes.
• Combining PICNIC with a truncated crRNA allows PAM-independent detection of single-nucleotide polymorphisms.
• The method successfully detected a drug-resistant HIV-1 K103N mutant and genotyped HCV variants in clinical samples with 100% specificity.

Conclusions:

• PICNIC significantly enhances the flexibility and precision of CRISPR-Cas12 diagnostics.
• The method overcomes the PAM sequence restriction, broadening the scope of CRISPR-based detection.
• PICNIC offers a simple yet groundbreaking solution for advanced molecular diagnostics.