Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Point-of-care nucleic acid diagnostics are crucial for overcoming laboratory-based scaling limitations.
• Isothermal amplification combined with CRISPR-based detection offers rapid, programmable pathogen testing solutions.

Purpose of the Study:

• To develop and optimize a one-pot NASBA-Cas13a nucleic acid detection assay for point-of-care pathogen testing.
• To establish design rules for NASBA primers and LbuCas13a guide RNAs for sensitive viral RNA detection.
• To investigate the combination of high-throughput screening and mechanistic modeling for system understanding and optimization.

Main Methods:

• Developed an in-house formulation of Nucleic Acid Sequence-Based Amplification (NASBA) for isothermal amplification.
• Designed NASBA primer sets and LbuCas13a guide RNAs for SARS-CoV-2 RNA detection.
• Employed high-throughput assay condition screening and ordinary differential equation modeling.

Main Results:

• Achieved 20-200 aM sensitivity for SARS-CoV-2 viral RNA fragments using the NASBA-Cas13a assay.
• Demonstrated a sensitive and fast nucleic acid detection method without requiring specialized equipment.
• Gained a mechanistic understanding of the NASBA-Cas13a reaction scheme through integrated experimental and modeling approaches.

Conclusions:

• The developed one-pot NASBA-Cas13a assay provides a framework for rapid, sensitive, and programmable point-of-care pathogen diagnostics.
• The study presents a valuable approach for understanding and optimizing nucleic acid detection systems.
• This work is anticipated to advance the development of future nucleic acid detection technologies.