Methods for Cas13a expression and purification for use in CRISPR diagnostics

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Emerging infectious diseases, like COVID-19 caused by SARS-CoV-2, underscore the need for rapid and accurate diagnostic tools.
• Conventional nucleic acid diagnostic methods, such as qPCR, are sensitive and accurate but often resource-intensive and lab-bound.
• There is a growing demand for low-cost, rapid, point-of-care diagnostic solutions.

Purpose of the Study:

• To describe a detailed method for the expression, purification, and validation of Leptotrichia buccalis (Lbu) Cas13a.
• To facilitate the widespread adoption of LbuCas13a as a tool for molecular diagnostics.
• To enhance RNA detection capabilities for emerging infectious diseases.

Main Methods:

• Expression and purification of the LbuCas13a protein.
• Validation of LbuCas13a for RNA detection in complex biological samples.
• Characterization of LbuCas13a's performance with and without pre-amplification.

Main Results:

• Successfully expressed and purified functional LbuCas13a.
• Validated LbuCas13a for sensitive and rapid RNA detection.
• Demonstrated LbuCas13a's utility in complex mixtures, applicable to point-of-care diagnostics.

Conclusions:

• LbuCas13a is a potent CRISPR-based tool for rapid RNA detection.
• The described protocol enables the production and validation of LbuCas13a for diagnostic applications.
• This work supports the development of advanced point-of-care diagnostics for infectious diseases.