Co-freezing localized CRISPR-Cas12a system enables rapid and sensitive nucleic acid analysis

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostics

Background:

• Sensitive nucleic acid detection is crucial for disease diagnosis and treatment monitoring.
• Existing localized CRISPR-Cas12a strategies require optimization for speed and efficiency.

Purpose of the Study:

• To develop a rapid and sensitive nucleic acid detection strategy using a co-freezing localized CRISPR-Cas12a (CL-Cas12a) approach.
• To evaluate the performance of CL-Cas12a for detecting specific nucleic acid targets and imaging in living cells.

Main Methods:

• A 15-minute co-freezing process was employed to create the CL-Cas12a complex, immobilizing Cas12a/crRNA and hairpin reporters on gold nanoparticles (AuNPs).
• The CL-Cas12a platform was utilized for quantitative nucleic acid detection, specificity testing, and application in detecting human papillomavirus and lung cancer mutations.
• Signal amplification techniques were used for imaging Survivin mRNA in living cells.

Main Results:

• The CL-Cas12a strategy achieved highly efficient loading of components on AuNPs, leading to rapid sensing.
• Quantitative detection of nucleic acid targets down to 98 aM was achieved within 30 minutes with excellent specificity.
• Successful application in detecting human papillomavirus infection, lung cancer mutations, and imaging mRNA in living cells was demonstrated.

Conclusions:

• The co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy provides a rapid, sensitive, and specific method for nucleic acid detection.
• CL-Cas12a shows significant potential as an effective tool for clinical diagnostics, disease monitoring, and biological imaging.