PAM-Independent CRISPR-Cas12a System for Specific Assays of Single Nucleotide Variants

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• CRISPR-Cas12a is a sensitive nucleic acid detection tool.
• Its reliance on protospacer adjacent motifs (PAM) limits its diagnostic applications.
• Overcoming PAM dependency is crucial for broader CRISPR utility.

Purpose of the Study:

• To develop a protospacer adjacent motif (PAM)-independent CRISPR-Cas12a system.
• To enhance the flexibility and accessibility of CRISPR-based molecular diagnostics.
• To create a robust and high-fidelity nucleic acid detection assay.

Main Methods:

• Development of the TRACER (mutant target-recognized PAM-independent CRISPR-Cas12a enzyme reporting system).
• Utilizing a modified CRISPR-Cas12a enzyme for PAM-independent target recognition.
• Assessing sensitivity and specificity using low-concentration nucleic acid targets and single nucleotide variants (SNVs).

Main Results:

• TRACER achieves highly sensitive detection of nucleic acids down to 0.5 aM.
• It can identify single nucleotide variants (SNVs) at 0.1% in heterozygotes.
• TRACER demonstrates comparable accuracy to Sanger sequencing for clinical tumor samples.

Conclusions:

• TRACER offers a novel, PAM-independent approach for single nucleotide variant (SNV) assays.
• This system significantly broadens the application scope of CRISPR-Cas12a.
• TRACER holds substantial potential for clinical diagnostics, biomedical research, and drug discovery.