Fast and sensitive CRISPR detection by minimized interference of target amplification

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• CRISPR-based detection holds promise but lags behind existing diagnostics for point-of-care applications.
• Key limitations in Cas12b and Cas13a enzyme performance hinder their diagnostic utility.
• Interference during amplification and target degradation reduces sensitivity and efficiency.

Purpose of the Study:

• To identify and address rate-limiting factors in Cas12b and Cas13a CRISPR detection systems.
• To enhance the sensitivity and efficiency of CRISPR-based diagnostics for pathogen detection.
• To develop competitive CRISPR assays for on-site and in-home testing.

Main Methods:

• Engineered a protospacer-adjacent motif-interacting domain in Cas12b to improve one-pot testing sensitivity.
• Optimized Cas13a assay conditions to minimize interference with viral RNA during amplification.
• Evaluated assay performance using clinical samples for SARS-CoV-2 detection.

Main Results:

• Engineered Cas12b demonstrated 10–10,000-fold improved sensitivity, detecting SARS-CoV-2 at 0.5 cp μl⁻¹ in 100% of clinical samples.
• Optimized Cas13a assay achieved 0.5 cp μl⁻¹ sensitivity for SARS-CoV-2 detection in 30 minutes at room temperature, with 98.8% accuracy.
• Both optimized CRISPR systems exhibited enhanced performance and sensitivity compared to wild-type counterparts.

Conclusions:

• CRISPR-based detection, particularly with engineered Cas12b and optimized Cas13a, is now competitive with established diagnostic methods.
• Relaxed reaction conditions and improved performance enable widespread application of CRISPR diagnostics for pathogen surveillance.
• These advancements pave the way for rapid, sensitive, and accessible on-site and in-home diagnostic testing.