Methods for Cas13a expression and purification for use in CRISPR diagnostics.

Abstract

The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.