Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• CRISPR-Cas13 systems offer unique collateral activity for nucleic acid diagnostics.
• Engineering Cas13 for enhanced collateral activity is challenging due to complex structural dynamics.
• Optimizing Cas13 systems is crucial for advancing nucleic acid detection sensitivity.

Purpose of the Study:

• To engineer Leptotrichia wadei Cas13a (LwaCas13a) with enhanced collateral activity.
• To improve the sensitivity of Cas13a-based nucleic acid detection.
• To develop a preamplification-free diagnostic method for pathogens like SARS-CoV-2.

Main Methods:

• Engineered LwaCas13a by inserting RNA-binding domains into an active-site-proximal loop.
• Assessed collateral activity and sensitivity of engineered LwaCas13a variants.
• Integrated engineered variants with an electrochemical method for SARS-CoV-2 detection.

Main Results:

• Two LwaCas13a variants exhibited significantly enhanced collateral activity and sensitivity compared to wild-type.
• Detected SARS-CoV-2 genome at attomolar concentrations in inactive viral and unextracted clinical samples.
• Achieved ultrasensitive detection without the need for target preamplification.

Conclusions:

• Engineered LwaCas13a variants possess superior collateral activity for nucleic acid detection.
• The enhanced LwaCas13a enzymes enable highly sensitive, preamplification-free diagnostics.
• These engineered enzymes are suitable for integration into various Cas13a-based detection platforms.